Gli Proteins Research Articles

Abstract 5283: Characterization of Gli activation by the estrogen receptor in breast cancer cells

Abstract Background: The nuclear steroid receptor superfamily encompasses a group of proteins best known fortheir functions as primary transcription factors that are conditionally active when bound to a ligand.Here, we show that a prominent member of this family, the estrogen receptor [ER-α] have a secondaryfunction of activating the Gli family of transcription factors. Gli is recognized as the mediator of activeHedgehog (Hh) signaling and plays an important role in cell development and growth. Gli activity isregulated by a post-translational proteolytic process that is suppressed by Hedgehog signaling.Previously we found that in prostate cancer, the ligand activated androgen receptor [AR] recognizes andbinds to Gli proteins at their Protein Processing Domains. This binding stabilizes Gli proteins in their un-proteolyzed active form and bypasses the Hedgehog signaling, thus promoting cancer progressionthrough non-canonical activation of Gli proteins. Due to the high similarity between AR and ER, wehypothesized that a similar Gli regulation could play a role in Breast Cancer (BrCA) progression. We thustested the ability of human ER-α to bind and activate Gli in BrCa and evaluated the role of this pathwayin tumor cell growth. Methods: we measured Gli activity in 293T and BrCa cells (MCF7, T47D, MDA-MB-453) in presence andabsence of steroid ligand using Gli-luciferase reporter assay. We evaluated the interaction between Gli3and ER-α by co-immunoprecipitation and proximity ligation assay and assessed the stability of Gli3stability in BrCa cell extracts by western blots. We also studied the effect of ER-α knockdown ordestabilization (by fulvestrant treatment) on Gli3 stability, formation of intranuclear ER-α-Gli3complexes and Gli reporter activity. We also measured the expression level of Gli target genes in thepresence and absence of estradiol by qPCR in BrCa cells. Lastly, we evaluated the importance of Gli3expression on BrCa growth by Gli3 knockdown and Cyquant assay. Results: We found that ER co-immunoprecipitates with Gli3. Transfection with ER-α increased Glireporter activity which was further increased by estradiol treatment. Acute (2hr) estradiol treatmentincreased intranuclear ER-α-Gli3 complex formation in BrCa cells. Chronic (48hr) estradiol treatmentincreased Gli3 stability and endogenous activity in BrCa cells. Destabilization or knockdown of ER-αdecreased estradiol-induced formation of ER-α-Gli3 complexes as well as Gli activity and stability in BrCacells. In addition, siRNA knockdown of Gli3 reduced growth in BrCa cells. Conclusion: Collectively our results uncovered a new role of the steroid receptors ER and AR inregulating Gli oncogenic transcriptional activity in BrCa and PCa, respectively. Citation Format: Shabnam Massah, Jane Foo, Na Li, Sarah Truong, Mannan Nouri, Lishi Xie, Gail Prins, Ralph Buttyan, Nada Lallous, Artem Cherkasov. Characterization of Gli activation by the estrogen receptor in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5283.

Abstract 2305: Centriolin depletion inhibits cellular proliferation of rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, accounts for approximately five percent of all childhood cancers. It develops from rhabdomyoblasts and can arise in a variety of anatomic locations. Recent findings have assisted the prediction of disease prognosis and treatment regimens based on genetic and cellular differences among the major types of RMS. One potential target is a cellular appendage called the primary cilium. Primary cilia are thought to play a critical role in the regulation of myogenesis, a process deregulated in RMS. During myogenesis, Gli protein accumulates at the primary cilia in myoblasts and induces proliferation. The centrosome protein, centriolin (CNTRL), is known to be a key structural component of primary cilia. Therefore, we hypothesized that disruption of CNTRL, using CRISPR/Cas9, may affect Gli protein function and, therefore, the proliferation of these cells. Two cell lines were used: A204, a rhabdoid cell line, and CCL-136, an embryonal RMS cell line. When CNTRL was depleted and analyzed 2 days post-transfection, a significant reduction in cell viability was observed in CCL-136 compared to control, whereas no significant increase in cell death in A204 was observed. Gli1 expression decreased in both cells lines when CNTRL was eliminated, while cell cycle progression was also reduced, as confirmed by immunofluorescence staining of Ki67. Unexpectedly, primary cilia formation, indicated by Arl13B expression, increased in A204 with CNTRL loss, with no perceptible change in CCL-136 cells. To assess differentiation potential, A204 and CCL-136 were cultured in differentiation media using 2% horse serum. Separately, cells were cultured in 0% FBS (serum) to test differentiation potential under starvation conditions. As expected, both cell lines expressed robust levels of MyoG, a muscle differentiation marker, with 2% horse serum. However, loss of CNTRL leads to a perceptible decrease in MyoG in the CCL-136, and a slight decrease in A204. Additionally, wound healing assays were conducted and showed CCL-136 had inherently greater ability to migrate 24 hours post-wound compared to A204., However, CCL-136 showed diminished migration under stressed conditions. Interestingly, cell migration was significantly diminished in A204 under starvation conditions when CNTRL was depleted (p<0.05). We show that CNTRL loss inhibits the cell cycle in two different forms of RMS. This arrest occurs with a corresponding reduction in Gli1 expression. This confirms previous studies that suggest Gli1 is a critical factor in maintaining the proliferative state. In addition, our results show that in cells normally producing primary cilia, like A204, CNTRL loss increases the number of ciliated cells. Taken together, our data support the possibility of inhibiting cellular proliferation of RMS cells through manipulation of CNTRL and suggest that CNTRL may be a viable future treatment target. Citation Format: Yoshinori Takeda, Ariel Cole, Caitlin Boyle, Adam Gromley. Centriolin depletion inhibits cellular proliferation of rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2305.

Expression of tumour transcription factor GLI1 in canine mammary tumours tissue.

BackgroundMammary tumor is one of the most common diseases of canine in pet clinics.ObjectivesThis study investigates the distribution and expression of the tumor transcription factor GLI1 and the downstream proteins, Bmi1 and Sox2, in canine mammary tumors and paracancerous tissues.MethodsCancerous and paracancerous normal mammary tissues were detected using western blotting (WB), and immunohistochemistry.ResultsThe results showed that the histopathology of different types in mammary tumors by microscopic observation. GLI1/Bmi1/Sox2 expression was significantly higher in canine mammary invasive carcinoma than in ductal carcinoma and adjacent normal mammary tissues (p < 0.01). The expression of GLI1 in invasive carcinoma tissues was significantly higher than Bmi1 and Sox2, while Sox2 expression in ductal carcinoma tissues was significantly higher than GLI1 and Bmi1 (p < 0.01). GLI1/Bmi1/Sox2 all showed positive reactions in both mammary tumor and adjacent normal mammary tissues with immunohistochemistry. GLI1 and Sox2 showed strong positive staining in the cytoplasm of invasive mammary carcinoma and ductal carcinoma cells, and weak positive staining in the nuclei. The positive Bmi1 reaction was mainly concentrated in the cytoplasm of invasive carcinoma and ductal carcinoma cells, while the positive reaction on the cell membrane was weak.ConclusionsWe speculate that GLI1 and related proteins play an important role in regulating the proliferation and differentiation of tumors. Therefore, it provides important reference for the pathogenesis and pathogenicity of canine mammary tumor.

Indolactam Dipeptides as Nanomolar Gli Inhibitors.

The Gli transcription factors within the Hedgehog (Hh) signaling pathway play essential roles in human development. However, the reactivation of Gli proteins in adult tissue is tumorigenic and drives the progression of several cancers, including the majority of basal cell carcinomas. Here we describe a novel set of indolactam dipeptides that target protein kinase C (PKC), exploiting the unique capacity of PKC isozymes to act as regulators of Gli. We devised an efficient synthetic route for the indolactam-based natural product (-)-pendolmycin and a series of analogues, and we evaluated these analogues in mechanistically distinct Gli reporter assays. The lead compound from these studies, N-hexylindolactam V, exhibits superior Gli suppression relative to clinical inhibitors and blocks the growth of Gli-dependent basal cell carcinoma cells. More broadly, our structure-activity studies provide inroads for the development of novel Gli antagonists and new avenues for combating Gli-driven cancers.

The ciliary gene INPP5E confers dorsal telencephalic identity to human cortical organoids by negatively regulating Sonic hedgehog signaling.

Defects in primary cilia, cellular antennas that control multiple intracellular signaling pathways, underlie several neurodevelopmental disorders, but it remains unknown how cilia control essential steps in human brain formation. Here, we show that cilia are present on the apical surface of radial glial cells in human fetal forebrain. Interfering with cilia signaling in human organoids by mutating the INPP5E gene leads to the formation of ventral telencephalic cell types instead of cortical progenitors and neurons. INPP5E mutant organoids also show increased Sonic hedgehog (SHH) signaling, and cyclopamine treatment partially rescues this ventralization. In addition, ciliary expression of SMO, GLI2, GPR161, and several intraflagellar transport (IFT) proteins is increased. Overall, these findings establish the importance of primary cilia for dorsal and ventral patterning in human corticogenesis, indicate a tissue-specific role of INPP5E as a negative regulator of SHH signaling, and have implications for the emerging roles of cilia in the pathogenesis of neurodevelopmental disorders.

INVESTIGASI BAHAN AKTIF PEGAGAN BERDASARKAN JEJARING DENGAN PROTEIN TARGET : STUDI PENCARIAN OBAT ALZHEIMER SECARA IN SILICO

&#x0D; ABSTRACT&#x0D; Alzheimer’s disease is a top-10 deadly neurodegenerative disease based on WHO in 2019. Its characterized by a reduction in Choline Acetyltransferase (ChAt) of a substance that acts in the production of asetikolin. One of the ingredients of nature known to play a part in the increased memory of pegagan. To learn about the line of active compound mechanisms used in the treatment of alzheimer's, the study uses an in silico approach by analyzing target proteins and the compound's active compound on a network of compounds. This study used 12 test compounds and the ChAt receptor molecules. Networking design the interactions of these compounds using the sea target and STRING databases. The results of that interactions are visualized on the cytoscape device. Furthermore, the results also suggested thateight of compounds’stest that have interactions with ChAt receptors whereas the target proteins directly linked to ChAt receptors namely Tryptophan 5-hydroxylase 1 (PDB ID: 5tpg) and Zinc finger protein GLI (PDB ID: 4kmd). Thus, the pegagan active compound that acts most closely on protein target is Asiatic Acid, Brahmic Acid, -Humulene, -Caryophyllene, Bicyclogermacrene, Germacrene B, -Pinene, Caryophyllene.&#x0D; Keywords: Alzheimer's disease, Choline Acetyltransferase (ChAt), pegagan, in silico, network interaction

Variants in genes related to development of the urinary system are associated with Mayer\u2013Rokitansky\u2013K\xfcster\u2013Hauser syndrome

Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome, also known as Müllerian agenesis, is characterized by uterovaginal aplasia in an otherwise phenotypically normal female with a normal 46,XX karyotype. Previous studies have associated sequence variants of PAX8, TBX6, GEN1, WNT4, WNT9B, BMP4, BMP7, HOXA10, EMX2, LHX1, GREB1L, LAMC1, and other genes with MRKH syndrome. The purpose of this study was to identify the novel genetic causes of MRKH syndrome. Ten patients with MRKH syndrome were recruited at Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China. Whole-exome sequencing was performed for each patient. Sanger sequencing confirmed the potential causative genetic variants in each patient. In silico analysis and American College of Medical Genetics and Genomics (ACMG) guidelines helped to classify the pathogenicity of each variant. The Robetta online protein structure prediction tool determined whether the variants affected protein structures. Eleven variants were identified in 90% (9/10) of the patients and were considered a molecular genetic diagnosis of MRKH syndrome. These 11 variants were related to nine genes: TBC1D1, KMT2D, HOXD3, DLG5, GLI3, HIRA, GATA3, LIFR, and CLIP1. Sequence variants of TBC1D1 were found in two unrelated patients. All variants were heterozygous. These changes included one frameshift variant, one stop-codon variant, and nine missense variants. All identified variants were absent or rare in gnomAD East Asian populations. Two of the 11 variants (18.2%) were classified as pathogenic according to the ACMG guidelines, and the remaining nine (81.8%) were classified as variants of uncertain significance. Robetta online protein structure prediction analysis suggested that missense variants in TBC1D1 (p.E357Q), HOXD3 (p.P192R), and GLI3 (p.L299V) proteins caused significant structural changes compared to those in wild-type proteins, which in turn may lead to changes in protein function. This study identified many novel genes, especially TBC1D1, related to the pathogenesis of MRKH syndrome. The identification of these variants provides new insights into the etiology of MRKH syndrome and a new molecular genetic reference for the development of the reproductive tract.

Delivery of Targeted Co(III)-DNA Inhibitors of Gli Proteins to Disrupt Hedgehog Signaling.

The Hedgehog (Hh) signaling pathway is integral for embryonic development and normal cell maintenance. However, aberrant expression of the Hh pathway is recognized as the oncogenic driving force for basal cell carcinoma (BCC). Current chemotherapeutic treatments that inhibit Hh signaling allow treatment of only locally advanced and metastatic BCCs via inhibition of the transmembrane protein, smoothened. It is further recognized that downstream mutations often lead to chemoresistant tumor recurrence. The Gli proteins are the ultimate regulators of Hh signaling and belong to a family of Cys2His2 zinc finger transcription factors (ZnFTFs) that we have shown can be irreversibly inhibited by a series of cobalt(III) Schiff base-DNA (CoSB-DNA) conjugates. However, a significant challenge is the delivery of CoSB-DNA complexes in mammalian tissues. Herein, we report a polyethyleneimine-functionalized graphene oxide nanoconjugate (GOPEI) that delivers CoGli, a CoSB-DNA complex that targets Gli specifically. We describe the characterization of the surface functionalization of GOPEI and accumulation in ASZ murine BCC cells via confocal microscopy and inductively coupled plasma-mass spectrometry (ICP-MS). Lysosomal escape of CoGli is further confirmed by confocal microscopy. We report the successful targeting of Gli by CoGli and a 17-fold improvement in potency over small-molecule Gli inhibitor GANT-61 in inhibiting Hh-driven migration of ASZ murine BCC cells. This study provides a promising starting point for further investigating CoGli inhibitors of Hh signaling in developed mammalian tissues.

Two nonsense GLI3 variants are associated with polydactyly and syndactyly in two families by affecting the sonic hedgehog signaling pathway.

BackgroundPolydactyly and syndactyly are congenital limb deformities, segregating in an autosomal‐dominant fashion. The variants in the GLI3 gene are closely related to congenital limb malformations. However, the causes underlying polydactyly and syndactyly are not well understood.MethodsWe conducted a whole‐exome sequencing on two four‐generation Chinese families with polydactyly and syndactyly. Then c.2374C>T and c.1728C>A mutant plasmids were transfected to HEK293T cells and mice limb bud cells to explore the functional consequences of these variants. Western blot and real‐time quantitative PCR were used to analyze the expression of GLI3 and Shh.ResultsIn these two families, the known GLI3 variant (NM_000168.6:c.2374C>T) and the novel GLI3 variant (NM_000168.6:c.1728C>A) contributed to polydactyly and syndactyly. Additionally, the GLI3 c.2374C>T mutant plasmid led to truncated GLI3 protein, and the GLI3 c.1728C>A mutant plasmid led to degraded GLI3 protein. Simultaneously, we demonstrated that the GLI3‐mutant plasmids led to decreased Shh expression in mice limb bud cells.ConclusionWe demonstrated that the novel GLI3 variant (c.1728C>A) and known GLI3 variant (c.2374C>T) contributed to the malformations in two four‐generation pedigrees with polydactyly and syndactyly by affecting SHH signaling.

Cepharanthine hydrochloride inhibits the Wnt/β‑catenin/Hedgehog signaling axis in liver cancer.

Cepharanthine, a biscoclaurine alkaloid isolated from the roots of Stephania cephalantha Hayata, has been reported to demonstrate antitumor activity across multiple cancer types; however, the mechanisms are still under investigation. High transcriptional responses by both the Hedgehog and Wnt pathways are frequently associated with specific human cancers, including liver cancer. To investigate whether these signaling pathways are involved in the pharmaceutical action of cepharanthine, we investigated Hedgehog and Wnt signaling in models of liver cancer treated with a semi-synthetic cepharanthine derivative, cepharanthine hydrochloride (CH), in vitro and in vivo. By using MTT cytotoxic, scratch, Transwell, colony formation and flow cytometry assays, the pharmaceutical effect of CH was assessed. The compound was found to inhibit cellular proliferation and invasion, and promote apoptosis. Subsequent mechanistic investigations revealed that CH suppressed the Hedgehog/Gli1 signaling pathway by inhibiting Gli1 transcription and its transcriptional activity. CH also inhibited Wnt/β-catenin signaling, and the pathway was found to be an upstream regulator of Hedgehog signaling in CH-treated liver cancer cells. Finally, the antitumor effects of CH were demonstrated in an in vivo xenograft tumor model. Immunohistochemical analysis indicated that Gli1 protein levels were diminished in CH-treated xenografts, compared with that noted in the controls. In summary, our results highlight a novel pharmaceutical antitumor mechanism of cepharanthine and provide support for CH as a clinical therapy for refractory liver cancer and other Wnt/Hedgehog-driven cancers.

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

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

GLI transcriptional repression is inert prior to Hedgehog pathway activation

The Hedgehog (HH) pathway regulates a spectrum of developmental processes through the transcriptional mediation of GLI proteins. GLI repressors control tissue patterning by preventing sub-threshold activation of HH target genes, presumably even before HH induction, while lack of GLI repression activates most targets. Despite GLI repression being central to HH regulation, it is unknown when it first becomes established in HH-responsive tissues. Here, we investigate whether GLI3 prevents precocious gene expression during limb development. Contrary to current dogma, we find that GLI3 is inert prior to HH signaling. While GLI3 binds to most targets, loss of Gli3 does not increase target gene expression, enhancer acetylation or accessibility, as it does post-HH signaling. Furthermore, GLI repression is established independently of HH signaling, but after its onset. Collectively, these surprising results challenge current GLI pre-patterning models and demonstrate that GLI repression is not a default state for the HH pathway.

Zic5 stabilizes Gli3 via a non-transcriptional mechanism during retinal development.

The Zic family of zinc finger transcription factors plays a critical role in multiple developmental processes. Using loss-of-function studies, we find that Zic5 is important for the differentiation of retinal pigmented epithelium (RPE) and the rod photoreceptor layer through suppressing Hedgehog (Hh) signaling. Further, Zic5 interacts with the critical Hh signaling molecule, Gli3, through the zinc finger domains of both proteins. This Zic5-Gli3 interaction disrupts Gli3/Gli3 homodimerization, resulting in Gli3 protein stabilization via a reduction in Gli3 ubiquitination. During embryonic Hh signaling, the activator form of Gli is normally converted to a repressor form through proteosome-mediated processing of Gli3, and the ratio of Gli3 repressor to full-length (activator) form of Gli3 determines the Gli3 repressor output required for normal eye development. Our results suggest Zic5 is a critical player in regulating Gli3 stability for the proper differentiation of RPE and rod photoreceptor layer during Xenopus eye development.

The Effects of Hedgehog Signaling Pathway on the Proliferation and Apoptosis of Melanoma Cells.

Background Studies have found that the abnormality of the Hedgehog signaling pathway is related to the occurrence and development of a variety of tumors, but the effect of this signaling pathway on melanoma cells is still unclear. Methods This study aimed to discuss the effect of Hedgehog signaling pathway on the proliferation and apoptosis of human malignant melanoma A375 cells and explore its possible mechanism in the proliferation and apoptosis of melanoma cells. Different concentrations of Hedgehog signaling pathway inhibitor cyclopamine (5, 10, 20 and 40 μM) were used to treat human melanoma A375 cells for 24, 48, and 72 h, and set a blank control group (0 μM). Trypan blue cell counting method was used to detect cell viability. MTT method was used to detect the inhibition rate of cell proliferation. Transwell was used to detect cell invasion, and flow cytometry was used to detect cell apoptosis. Results Through the trypan blue cell counting method and MTT experiment, it was found that the Hedgehog signaling pathway inhibitor cyclopamine has an inhibitory effect on the proliferation and viability of melanoma A375 cells (P < 0.05), and the proliferation inhibitory effect is enhanced with prolonged action time in a dose- and time-dependent manner. Transwell experiment showed that compared with the blank control group, the invasion and migration ability of the treated melanoma A375 cells are significantly reduced, and the difference is statistically significant (P < 0.05). Cell apoptosis experiment showed that compared with the blank control group, the apoptosis rate of A375 cells is significantly higher after treated by 40 μM cyclopamine for 24 h, and the difference is statistically significant (P < 0.05). Gli1 and Bcl-2 protein are highly expressed in melanoma A375 cells, and their expressions show a downward trend (P < 0.05) after being treated by cyclopamine. Conclusion Cyclopamine inhibits cell proliferation and induces cell apoptosis by downregulating Gli1. Hedgehog signaling pathway can be used as a new target for the treatment of malignant melanoma, and multiple measures can be used to inhibit the signaling pathway to achieve a therapeutic effect.

The transcript ENST00000444125 of lncRNA LINC01503 promotes cancer stem cell properties of glioblastoma cells via reducing FBXW1 mediated GLI2 degradation

LINC010503 is a novel oncogenic lncRNA in multiple cancers. In this study, we further explored the expression of LINC010503 transcripts and their regulations on the glioblastoma (GBM) stem cell (GSC) properties. LINC01503 transcription patterns in GBM and normal brain tissues were compared using RNA-seq data from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA)-GBM. GBM cell lines (U251 and U87) were used as in vitro cell models for cellular and molecular studies. The results showed that ENST00000444125 was the dominant transcript of LINC01503 in both normal and tumor tissues. Its expression was significantly elevated in the tumor group and associated with poor survival outcomes. LINC01503 had both cytoplasmic and nuclear distribution. It positively modulated the expression of multiple GSC markers, including CD133, SOX2, NESTIN, ALDH1A1, and MSI1, and tumorsphere formation in U251 and U87 cells. RNA pull-down and RIP-qPCR assay confirmed an interaction between ENST00000444125 and GLI2. ENST00000444125 positively regulated the half-life of the GLI2 protein in GBM cells. ENST00000444125 overexpression reduced GLI2 ubiquitination and partially attenuated FBXW1 overexpression induced GLI2 ubiquitination. ENST00000444125 overexpression could activate Wnt/β-catenin signaling in GBM cells. However, these activating effects were remarkedly hampered when GLI2 was knocked down. In conclusion, this study revealed that LINC01503 might have isoform-specific dysregulation in GBM. Among the two major transcripts expressed in GBM cells, ENST00000444125 might be the major functional transcript. Its upregulation might enhance the GSC properties of GBM cells via reducing FBXW1-mediated proteasomal degradation of GLI2.

Expression of Shh, Gli1, and Cyr61 in Gastric Cancer Predicts Overall Survival of Patients: A Retrospective Study.

This study aimed to evaluate the expression levels of Shh, Gli1, and Cyr61 proteins in gastric cancer tissues and analyze the relationship between these three proteins and the clinicopathological factors and prognosis of patients. This was a retrospective study. Four hundred gastric cancer tissue specimens from patients who underwent radical gastrectomy in Zhangye People's Hospital affiliated to Hexi University between February 2013 and February 2021 underwent immunohistochemical analysis. The positive expression rates of Shh, Gli1, and Cyr61 in gastric cancer tissues were 55.5%, 56.5%, and 64.5%, respectively. The expressions of Shh, Gli1, and Cyr61 in gastric cancer tissues were significantly correlated with tumor size, depth of invasion, and degree of differentiation (P < .05). The expression of Shh protein was positively correlated with the expression of Gli1 protein (P < .01), and the expression of Gli1 protein was positively correlated with the expression of Cyr61 protein (P < .01). Univariate and multivariate analyses showed that the expression of Shh, Gli1, and Cyr61 could predict the prognosis of patients (P < .05). Receiver operating characteristic curve analysis combined with TNM staging could better predict the three-year overall survival of patients (P < .05). Shh, Gli1, and Cyr61 proteins are significantly expressed in gastric cancer tissues and are risk factors for the prognosis of patients with gastric cancer.

The PROTAC selectively degrading Bcl-xL represents a novel Hedgehog pathway inhibitor with capacity of combating resistance to Smoothened inhibitors while sparing bone growth.

Rationale: Primary and acquired resistance to Smoothened (Smo) inhibitors largely hampered their clinical efficacy. Given the important functions of hedgehog (Hh) pathway in bone formation and development, the permanent defects in bone growth caused by Smo inhibitors further restrict the use of Smo inhibitors for pediatric tumor patients. Anti-apoptotic Bcl-2 proteins regulate Hh activity by engaging a Bcl-2 homology (BH) domain sequence found in suppressor of fused (Sufu). In this study, we tested the effect of SIAIS361034, a Proteolysis Targeting Chimera (PROTAC) specifically targeting B-cell lymphoma extra large (Bcl-xL) to the celeblon (CRBN) E3 ligase for degradation, on combating the resistance and reducing the toxicity of bone growth caused by Hh inhibition. Methods: Fluorescence polarization, homogeneous time-resolved fluorescence (HTRF) assay, immunoblot, and immunoprecipitation (IP) were used to evaluate whether SIAIS361034 is an appropriate Bcl-xL PROTAC. Dual luciferase reporter assay, real-time quantitative PCR (RT-qPCR), depilatory model, and SmoA1 model were established to assess the effect of SIAIS361034 on the activity of Hh signaling pathway and its ability to overcome drug resistance in vitro and in vivo. Molecular mechanisms of SIAIS361034 for inhibiting Hh activity were demonstrated by dual luciferase reporter assay, immunoblot, and immunofluorescence staining. PET-CT and histopathology of bone tissues were used to assess the effects of SIAIS361034 on bone growth. Results: We observed that SIAIS361034 efficiently and selectively inhibits the activity of the Hh pathway in vitro and in vivo, by interrupting Bcl-xL/Sufu interaction, therefore, promoting the interaction of Sufu with Gli1. Moreover, SIAIS361034 possesses the ability of combating resistance to current Smo inhibitors caused by Smo mutations and Gli2 amplification and remarkably inhibits the growth of SmoA1 tumors in vivo. In contrast to von Hippel-Lindau (VHL) E3 ligase, our result further reveals little detectable expression of CRBN in two types of cells critical for bone development, human articular chondrocytes and human fetal osteoblastic cells. Moreover, treatment with SIAIS361034 results in no impairment on the bone growth of young mice, accompanying no alteration of the expression of Bcl-xL and Gli1 proteins. Conclusion: Our findings demonstrate that selectively targeting Bcl-xL by PROTAC is a promising strategy for combating resistance to Smo inhibitors without causing on-target drug toxicities of bone growth.

Propofol Inhibits Thyroid Cancer Cell Proliferation, Migration, and Invasion by Suppressing SHH and PI3K/AKT Signaling Pathways via the miR-141-3p/BRD4 Axis.

Objective This study explores the effect and mechanism of propofol for thyroid tumor. Methods Culture human normal thyroid cells Nthy-ori 3-1 and thyroid cancer cell line TPC-1. TPC-1 cells were divided into the propofol group (treated with propofol), miR-141-3p group (transfected with the miR-141-3p mimic), negative control group (transfected with miR-NC), miR-141-3p + pcDNA-BRD4 group (transfected with the miR-141-3p mimic and pcDNA-BRD4), miR-141-3p + pcDNA group (transfected with the miR-141-3p mimic and pcDNA), siBRD4 group (transfected with siBRD4), and si-control group (transfected with si-control). The detection of miR-141-3p and BRD4 expression in cells was done by RT-qPCR, and the dual-luciferase reporter gene method and western blotting were used to verify the targeting relationship between miR-141-3p and BRD4. MTT method was used to test cell proliferation, transwell method was used to test cell migration and invasion, and western blotting was used to test SHH, GLI1, p-PI3K, and p-AKT protein expression. Results Compared with Nthy-ori 3-1 cells, the expression of miR-141-3p in TPC-1 cells was markedly decreased. Propofol treatment and excessive expression of miR-141-3p could influence the phenotype of TPC-1 cells. BRD4 is one of the target genes of miR-141-3p, and its expression is negatively regulated by miR-141-3p. Overexpression of BRD4 can partially reverse the restraining effect of miR-141-3p on the TPC-1 cell phenotype. Both miR-141-3p and BRD4 can regulate the activity of SHH and PI3K/AKT signaling pathways. Conclusion Propofol can inhibit the activity of SHH and PI3K/AKT pathways by targeting downregulating BRD4 through miR-141-3p, thereby inhibiting the phenotype of TPC-1 cells.

Electroacupuncture of "Shuigou"(GV26) improves neurological function by promoting angioge-nesis and Shh signaling in ischemic cerebral tissue of rats with cerebral infarction

To observe the effect of electroacupuncture (EA) of "Shuigou" (GV26) on the activities of sonic hedgehog(Shh) signaling molecules (Shh, Ptch, Smo，Gli and Gli2) in ischemic brain tissues in rats with cerebral ischemia (CI), so as to explore its mechanisms underlying improvement of CI. Male Wistar rats were randomly divided into blank control group(n=10), sham operation group (n=90), model group(n=90) and EA group (n=90). The CI model was established by occlusion of the right middle cerebral artery (MCAO). According to the postoperative time points of MCAO, the later three groups were further divided into 1, 3, 6, 9, 12 and 24 h, and 3, 7 and 12 d subgroups, with 10 rats in each subgroup. EA (15 Hz, 2 mA) was applied to GV26 for 20 min. The 1 h to 24 h subgroups were treated immediately after modeling, the 3-12 d subgroups treated one time a day. The neurological severity score (NSS, 0 to 18 points) was used to evaluate the rats' neurological function, and TTC staining was employed to assess the cerebral ischemic volume (percentage of cerebral infarct volume, CIV). Western blot was employed to detect the expression of Shh, Ptch, Smo, Gli1 and Gli2 proteins in the ischemic cerebral tissue. Compared with the sham operation group, the NSS scores of the model group increased at all time points (P<0.01). The percentages of CIV of the model group from 3 h to 12 d were obviously higher than those of the sham operation group (P<0.01). The NSS scores at 3, 7 and 12 d and the percentages of CIV at 1, 3, 7 and 12 d after MCAO were significant lower in the EA group than in the model group (P<0.05). The protein expression levels of Shh from 12 h to 12 d (i.e. 12 h, 24 h, 3, 7 and 12 d), Ptch from 6 h to 12 d, Smo from 9 h to 12 d, Gli1 at 9 h, 12 h, and from 3 d to 12 d, Gli2 at 6, 9 and 12 h, and 3 d were significantly higher in the model group than in the sham operation group (P<0.05, P<0.01), while those of Shh at 3, 7 and 12 d, Ptch from 24 h to 7 d, Smo from 12 h to 7 d, Gli1 from 24 h to 7 d, Gli2 at 12 h, 3 and 7 d were significantly higher in the EA group than in the model group (P<0.05, P<0.01). No statistical significances were found between the sham operation and the blank control groups in all the indexes mentioned above (P>0.05). EA of GV26 can improve neurological function and reduce infarct volume in MCAO rats, which may be related to its function in up-regulating the activities of Shh signaling pathway in the ischemic cerebral tissues.

SARI inhibits growth and reduces survival of oral squamous cell carcinomas (OSCC) by inducing endoplasmic reticulum stress

AimsSARI (suppressor of activator protein (AP)-1, regulated by interferon (IFN) was identified as a novel tumor suppressor by applying subtraction hybridization to terminally differentiating human melanoma cells. The anti-tumor activity of SARI and the correlation between expression and cancer aggression and metastasis has been examined in multiple cancers, but its potential role in oral squamous cell carcinomas (OSCC) has not been explored. MethodsSARI expression was monitored in tumor tissues of OSCC patients by performing immunohistochemistry. Ectopic expression of SARI was achieved using a replication defective adenovirus expressing SARI (Ad.SARI). A nude mouse xenograft model was used to evaluate the in vivo efficacy of SARI. Endoplasmic reticulum (ER) stress was monitored in SARI infected OSCC cells by confocal microscopy. Key findingIn this study, we demonstrate that SARI expression is significantly lower in OSCC tumor tissue as compared to normal adjacent tissue. Ectopic expression of SARI induces cancer-specific cell death in human OSCC cell lines and in a paclitaxel plus cisplatin non-responder OSCC patient-derived (PDC1) cell line. Mechanistically, SARI inhibits zinc finger protein GLI1 expression through induction of endoplasmic reticulum (ER) stress. Using a nude mouse xenograft model, we show that intratumoral injections of Ad.SARI significantly reduce PDC1 tumor burden, whereas treatment with an ER stress inhibitor efficiently rescues tumors from growth inhibition. SignificanceOverall, our data provides a link between induction of ER stress and inhibition of the GLI1/Hedgehog signaling pathway and the tumor suppressive activity of SARI in the context of OSCC.