Expression Levels Of Gli1 Research Articles

MDB-28. THE CYTOSKELETON REGULATORS FORMINS CONTROL THE SHH PATHWAY AND ARE INVOLVED IN MEDULLOBLASTOMA TUMORIGENESIS

Abstract BACKGROUND Medulloblastoma (MB) is a lethal pediatric malignancy of the cerebellum. Among MB’s molecular subgroups, Sonic Hedgehog (SHH) is the most abundant and it is characterized by alterations of key components of the SHH development pathway. However, the molecular mechanisms driving SHH-MB require to further be unveiled to design more effective therapies. Recently, we identified formins, well-known players of cytoskeletal dynamics, as regulators of SHH signaling able to affect SHH-MB growth. METHODS Luciferase functional assays, western-blot and RT-qPCR have been performed to analyze the effect of formins on GLI1 (the final effector of SHH signaling) transcriptional activity and expression. Proliferation assays and Brillouin microscopy have been carried out on primary murine SHH-MB cells to test the effect of formins on SHH-MB growth and stiffness. RESULTS We demonstrated that formins have a dual role in the regulation of SHH signaling, either positive or negative. The modulation of formins expression in SHH-dependent cell models affect GLI1 transcriptional activity and expression. Moreover, formins show opposite protein expression levels during the development of murine cerebellum, and their modulation impair the proliferation of granule cell progenitors (GCPs), the cells of origin of MB. Interestingly, formins protein levels are highly altered in SHH-MB samples. Notably, the overexpression or the genetic silencing of formins in SHH-MB primary cells from Math1-Cre/Ptc1fl/fl mice significantly impact on the cell proliferation as consequence of modulated GLI1 expression levels and the stiffness of primary SHH-MB cells, thus suggesting that formins could affect tumor cell motility and invasiveness. CONCLUSIONS Our findings identify formins as regulators of SHH signaling and illuminate on the role of cytoskeleton in SHH-MB for the design of innovative interventions.

GLI1 and PTTG1 expression in colorectal carcinoma patients undergoing radical surgery and their correlation with lymph node metastasis.

Few studies have investigated the expression of GLI1 and PTTG1 in patients undergoing radical surgery for colorectal carcinoma (CRC) and their association with lymph node metastasis (LNM). Therefore, more relevant studies and analyses need to be conducted. To explore GLI1 and PTTG1 expression in patients undergoing radical surgery for CRC and their correlation with LNM. This study selected 103 patients with CRC admitted to our hospital between April 2020 and April 2023. Sample specimens of CRC and adjacent tissues were collected to determine the positive rates and expression levels of GLI1 and PTTG1. The correlation of the two genes with patients' clinicopathological data (e.g., LNM) was explored, and differences in GLI1 and PTTG1 expression between patients with LNM and those without were analyzed. Receiver operating characteristic (ROC) curves were plotted to evaluate the predictive potential of the two genes for LNM in patients with CRC. Significantly higher positive rates and expression levels of GLI1 and PTTG1 were observed in CRC tissue samples compared with adjacent tissues. GLI1 and PTTG1 were strongly linked to LNM in patients undergoing radical surgery for CRC, with higher GLI1 and PTTG1 levels found in patients with LNM than in those without. The areas under the ROC curve of GLI1 and PTTG1 in assessing LNM in patients with CRC were 0.824 and 0.811, respectively. GLI1 and PTTG1 expression was upregulated in patients undergoing radical surgery for CRC and are significantly related to LNM in these patients. Moreover, high GLI1 and PTTG1 expression can indicate LNM in patients with CRC undergoing radical surgery. The expression of both genes has certain diagnostic and therapeutic significance.

Construction of a Lentiviral Vector for Fgfr2 Overexpression and its Impact on the Biological Behavior of Cranial Suture Mesenchymal Stem Cells.

Suture mesenchymal stem cells (SuSCs), possessing self-renewal and multilineage differentiation abilities, play a crucial role in cranial bone growth. However, the impact of the disease-causing fibroblast growth factor receptor 2 (FGFR2) mutation on SuSCs in Crouzon syndrome has not been explored. This study aims to employ a lentivirus to overexpress Fgfr2 and investigate its role in the pathogenesis of Crouzon syndrome. Starting with the prevalent FGFR2 mutation site in patients with Crouzon syndrome, a lentiviral vector carrying the Fgfr2.C361Y mutation was developed and transfected into SuSCs, with a determined multiplicity of infection values. The experimental group, SuSCs+Fgfr2.C361Y, was compared with the empty vector and normal SuSC groups. Cell proliferation, cycle, apoptosis, and osteogenic functionality were assessed using CCK-8 assays, flow cytometry, ALP activity assays, and real-time quantitative polymerase chain reaction. The lentiviral vector effectively infected SuSCs, leading to heightened Fgfr2 expression, with optimal multiplicity of infection values of 80. The experimental group demonstrated decreased proliferation activity and a higher apoptosis rate compared with controls (P < 0.05). After osteogenic induction, the experimental group showed significantly higher ALP activity than controls (P < 0.05). Real-time quantitative polymerase chain reaction indicated lower mRNA expression levels of Gli1, Axin2, Pcna, Cdk2, and Bcl-2 in the experimental group than controls, whereas Bax, Runx2, and Bmp-2 showed higher expression (P < 0.05). This study constructed a lentivirus vector to upregulate Fgfr2 expression in SuSCs, suppressing stem cell stemness by inhibiting proliferation, promoting apoptosis, and accelerating premature osteogenic differentiation, resulting in premature suture closure. These findings establish the groundwork for further understanding the pathogenesis of Crouzon syndrome.

En1 promotes cell proliferation and migration via Hedgehog signaling pathway in esophageal squamous cell carcinoma

Objective: To explore the function and mechanism of transcription factor En1 in esophageal squamous cell carcinoma (ESCC). Methods: The correlations of En1 with prognosis were analyzed using the overall survival data of 9 397 pan-cancer patients and progression-free survival data of 4 349 pan-cancer patients from The Cancer Genome Atlas (TCGA) database. The En1 expression data in 53 and 155 cases of ESCC and their paired adjacent tissues were from Gene Expression Omnibus (GEO) database and National Genomics Data Center-Genome Sequence Archive(NGDC-GSA)database. Lentivirus was used to generate En1 stable knockout cell lines KYSE180 and KYSE450. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The migration ability of the cells was detected by Transwell assay. The effect of En1 on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of En1, glioma-associated oncogene family zinc finger 1 (GLI1), glioma-associated oncogene family zinc finger 2 (GLI2) and smoothened (SMO). Results: Pan-cancer data from TCGA showed that patients with low En1 expression had longer overall survival and progression-free survival than patients with high En1 expression (P< 0.001). Data from GEO and GSA databases also showed a high expression level of En1 in ESCC tissues compared with paired tissues (P＜0.001). Proliferation was inhibited after knockout of En1 in KYSE180 and KYSE450 cells (P＜0.001). The colony formation numbers decreased. The colony formation numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 138.33±23.07 and 127.00±19.70, respectively, significantly lower than that of the shNC group 340.67±12.06 (P<0.001). The colony formation numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 65.33±2.52 and 9.00±3.00, respectively, significantly lower than that of the shNC group 139.00±13.00 (P<0.001). The migration numbers was inhibited after knockout of En1 [the Transwell numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 66.67±12.66 and 71.33±11.02, respectively, significantly lower than that of the shNC group 334.67±16.56 (P<0.001). The Transwell numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 112.33±14.57 and 54.33±5.51, respectively, significantly lower than that of the shNC group 253.33±21.03 (P<0.001)]. Xenograft model showed a slower growth rate of shEn1#1 and shEn1#2 cell lines (P＜0.001). The tumor weights of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were (0.046±0.026)g and (0.047±0.025)g, respectively, significantly lower than that of the shNC group (0.130±0.038)g (P＜0.001). After knockdown of En1, the relative expression levels of GLI1 in KYSE180 cells of the shEn1#1 group and the shEn1#2 group were 0.326±0.162 and 0.322±0.133, and the relative expression levels of GLI1 in KYSE450 cells of the shEn1#1 and shEn1#2 groups were 0.131±0.006 and 0.352±0.050, respectively, which were all lower than that in the shNC group (P＜0.01). After knockdown of En1, overexpression of GLI1 attenuated the inhibitory effect of knockdown of En1 on cell proliferation (P＜0.001), colony formation[the colony formation numbers of the shEn1#1-GLI1 group were 151.00±9.54, higher than 102.33±10.02 (P=0.004) of the shEn1#1-vector group] and migration [the migration numbers of the shEn1#1-GLI1 group were 193.67±10.07, higher than 109.33±11.50 (P<0.001) in the shEn1#1-vector group]. In clinical samples of ESCC, major regulatory factors of the Hedgehog pathway were up-regulated and the pathway was activated. Conclusion: En1 promotes the proliferation and migration of ESCC cells by regulating the Hedgehog pathway and can be used as a new potential target for targeted therapy of ESCC.

Increasing Sufu gene dosage reveals its unorthodox role in promoting polydactyly and medulloblastoma tumorigenesis.

Suppressor of fused (SUFU) is widely regarded as a key negative regulator of the sonic hedgehog (SHH) morphogenic pathway and a known tumor suppressor of medulloblastoma (MB). However, we report here that SUFU expression was markedly increased in 75% of specimens compiled in a tissue array comprising 49 unstratified MBs. The SUFU and GLI1 expression levels in this MB array showed strong positive correlation, which was also identified in a large public data set containing 736 MBs. We further report that increasing Sufu gene dosage in mice caused preaxial polydactyly, which was associated with the expansion of the Gli3 domain in the anterior limb bud and heightened Shh signaling responses during embryonic development. Increasing Sufu gene dosage also led to accelerated cerebellar development and, when combined with ablation of the Shh receptor encoded by Patched1 (Ptch1), promoted MB tumorigenesis. These data reveal multifaceted roles of SUFU in promoting MB tumorigenesis by enhancing SHH signaling. This revelation clarifies potentially counterintuitive clinical observation of high SUFU expression in MBs and may pave way for novel strategies to reduce or reverse MB progression.

Hedgehog signalling is involved in acquired resistance to KRASG12C inhibitors in lung cancer cells

Although KRASG12C inhibitors have shown promising activity in lung adenocarcinomas harbouring KRASG12C, acquired resistance to these therapies eventually occurs in most patients. Re-expression of KRAS is thought to be one of the main causes of acquired resistance. However, the mechanism through which cancer cells re-express KRAS is not fully understood. Here, we report that the Hedgehog signal is induced by KRASG12C inhibitors and mediates KRAS re-expression in cancer cells treated with a KRASG12C inhibitor. Further, KRASG12C inhibitors induced the formation of primary cilia and activated the Hedgehog-GLI-1 pathway. GLI-1 binds to the KRAS promoter region, enhancing KRAS promoter activity and KRAS expression. Inhibition of GLI using siRNA or the smoothened (Smo) inhibitor suppressed re-expression of KRAS in cells treated with a KRASG12C inhibitor. In addition, we demonstrate that KRASG12C inhibitors decreased Aurora kinase A (AURKA) levels in cancer cells, and inhibition of AURKA using siRNA or inhibitors led to increased expression levels of GLI-1 and KRAS even in the absence of KRAS inhibitor. Ectopic expression of AURKA attenuated the effect of KRASG12C inhibitors on the expression of GLI-1 and re-expression of KRAS. Together, these findings demonstrate the important role of AURKA, primary cilia, and Hedgehog signals in the re-expression of KRAS and therefore the induction of acquired resistance to KRASG12C inhibitors, and provide a rationale for targeting Hedgehog signalling to overcome acquired resistance to KRASG12C inhibitors.

The Relative Quantification of Hedgehog Signalling Components of Hepatocellular Carcinoma (Hepg-2) Cells Treated With Sorghum bicolor Leaf Stalk Ethanolic Extract

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer. Patients with advanced HCC have a dismal prognosis due to the limited therapy choices available. Hence, this present study evaluates the cytotoxic potential of the ethanolic crude extract of Sorghum bicolor leaf stalk (SBELSE) against human hepatocellular carcinoma (HEPG2) cell line, and its effect in the expression of its Hh signaling component-GLI1 and PTCH. The leaf stalk of Sorghum bicolor (SB) was pulverized and extracted with 70% ethanol. HEPG2 cells were treated with different concentration of ethanol extract of SBELSE which was subjected to 2-fold serial dilution to achieve a graded concentration (9.77-5000 µg/ml). Cell viability was performed using CCK-8 assay. The cells were treated with 8 µg/mL and 23 µg/mL concentration of SBELSE for 48 hour, after which RNA was isolated for downstream gene expression studies using RT-qPCR. The expression level of GLI1 and PTCH genes in cell line was examined relative to the untreated group. SBELSE exerted a cytotoxic effect on HEPG-2 cells with an inhibitory concentration (IC50) of 150 µg/mL. After 48 hours of treatment and evaluation, Expression of GLI1 and PTCH genes was seen in all cell group but was increased in groups treated with 8µg/mL concentration of S. bicolor extract than in the control group. The findings of this study suggest the possibility of the extract to contain agonistic component that might support cancer cell proliferation.

Solasodine suppress MCF7 breast cancer stem-like cells via targeting Hedgehog/Gli1

BackgroundRecently, a novel therapy to treat cancer has been to target cancer stem-like cells (CSCs). The aim of this study was to investigate the effect of solasodine, a steroidal alkaloid isolated from Solanum incanum L., on MCF7 CSCs and to understand the compound's underlying mechanism of action. MethodA tumorsphere formation assay was used to evaluate the effects of solasodine on the proliferation and self-renewal ability of MCF7 CSCs. The level of expression of proteins associated with cancer stemness markers and Hh signaling mediators was determined. The interaction between solasodine and Gli1 was calculated by molecular docking and further demonstrated by cellular thermal shift assay. ResultsSolasodine significantly decreased the proliferation of MCF7 tumorspheres and showed a stronger cytotoxicity on breast cancer cells with higher levels of Gli1 expression. The results showed that the levels of CD44 and ALDH1 expression were suppressed. Furthermore, expression of CD24 was enhanced by solasodine, via a mechanism that involved dampening Gli1 expression and blocking the nuclear translocation of this protein in MCF7 tumorspheres. Computational studies predicted that solasodine showed a high affinity with the Gli1 zinc finger domain that resulted from hydrogen-bonds to the THR243 and ASP216 amino acids residues. In addition, solasodine specifically bound with Gli1 and enhanced Gli1 protein stability in MCF7 cells. ConclusionHere, our findings indicated that solasodine can directly suppresses Hh/Gli1 signaling, and is a novel anticancer candidate that targets CSCs.

Effects of long-term exposure to MST-312 on lung cancer cells tumorigenesis: Role of SHH/GLI-1 axis.

Replicative immortality is a key feature of cancer cells and it is maintained by the expression of telomerase, a promising target of novel therapies. Long-term telomerase inhibition can induce resistance, but the mechanisms underlying this process remain unclear. The Sonic hedgehog pathway (SHH) is an embryogenic pathway involved in tumorigenesis and modulates the transcription of telomerase. We evaluated the effects of long-term treatment of the telomerase inhibitor MST-312 in morphology, proliferation, resistance, and in the SHH pathway molecules expression levels in lung cancer cells. Cells treated for 12 weeks with MST-312 showed changes in morphology, such as spindle-shaped cells, and a shift in the distribution of F-ACTIN from cortical to diffuse. Treatment also significantly reduced cells' efficiency to form spheroids and their clonogenic potential, independently of the cell cycle and telomeric DNA content. Moreover, GLI-1 expression levels were significantly reduced after 12 weeks of MST-312 treatment, indicating a possible inhibition of this signaling axis in the SHH pathway, without hindering NANOG and OCT4 expression. Here, we described a novel implication of long-term treatment with MST-312 functionally and molecularly, shedding new light on the molecular mechanisms of this drug in vitro.

Mechanism of Targeting the Hedgehog Signaling Pathway against Chemotherapeutic Resistance in Multiple Myeloma.

Objective The aim of this study was to explore the relationship between the Hedgehog signaling pathway and drug resistance in multiple myeloma. Methods The human myeloma cell line RPMI 8266 was taken as the research object. An azithromycin (AZM)-resistant cell line RPMI 8226/R was constructed, and GENT61 was used to block the Hedgehog signaling pathway. Cells were rolled into RPMI 8226/S (S group), RPMI 8226/R (R group), GENT61+RPMI 8226/S (GENT61+S group), and GENT61+RPMI 8226/R (GENT61+R group). The proliferation of cells in each group was assessed, and the expression of patched homolog 1 (PTCH1), zinc finger-containing transcription factors 1 (GLI1), GLI2, hair-division associated enhancer 1 (Hes1), and sonic hedgehog factor (SHH) in each group was detected. Interleukin (IL)-6 and vascular endothelial growth factor (VEGF) were measured. Results Compared with the S group, the expression levels of PTCH1, GLI2, Hes1, and SHH and the contents of IL-6 and VEGF in the R group were greatly increased, while the expression level of GLI1 was notably decreased (P < 0.05). Compared with the R group, the GENT61+R group greatly increased cell proliferation inhibition. However, the expression levels of PTCH1, GLI2, Hes1, and SHH, and the contents of IL-6 and VEGF were notably decreased, while GLI1 expression levels were greatly increased (P < 0.05). Conclusion AZM-resistant multiple myeloma was closely associated with the Hedgehog signaling pathway activation, and blocking the Hedgehog signaling pathway can be used as a therapeutic target to improve drug resistance in multiple myeloma.

Double immunohistochemistry of Gli-1 and cytokeratin-17 in ameloblastomas

<h3>Objective</h3> Our previous research showed relatively high RNA expression level of Gli1 present in ameloblastomas and frequently coexistent with <i>BRAF</i> mutation. Moreover, the mRNA expression level of Gli-1 seems to have positive correlation with acanthomatous change in the tumor nests morphologically. In order to investigate the relation- ship between Gli-1 and cytokeratin, double immunohistochemistry (IHC) of Gli-1 and cytokeratin -17 (CK17) was performed in ameloblastoma cases. <h3>Method</h3> Thirty formalin fixed paraffin embedded (FFPE) ameloblastoma tissue sections with known BRAFV600E status and Gli-1 mRNA expression level were included in the study. One syndrome associated odontogenic kerato- cyst was used as positive control and four radicular cysts and one calcifying odontogenic cyst as negative control. Double IHC of Gli-1 and CK17 was performed in these samples. Result: All of the 30 FFPE ameloblastoma cases revealed Gli-1 nuclear stain and CK17 cytoplasmic stain with various intensity and various amount of positive cells. Our results showed that Gli-1 expressed mainly in the peripheral ameloblast-like cells and the expression of CK17 was mainly in the central acanthomatous portion of the tumor nests. The expression of Gli-1 and CK17 was mainly not co-localized in the same tumor cells with few exceptional co-localized cells. In addition, the expression of CK17 was always present with Gli-1 positive cells at the periphery, and the Gli-1 negative tumor nests were not express CK17 in central. <h3>Conclusion</h3> Our results showed although Gli-1 mRNA expression was positively correlated with acanthomatous change in the tumor nests morphologically, Gli-1 and CK17 were not co-localized in majority of the cells, which indicated that Gli-1 may indirectly regulate the CK17 expression in ameloblastomas. The possible interpretation is that CK17 expression is a later event, which occurs after activation of SHH pathway.

Expression levels of sonic hedgehog pathway genes and their targets are upregulated in early clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH) molecular cascade is altered in various malignancies in tumorigenesis, and several SHH pathway inhibitors have been considered as potential anticancer drugs. The aim of the present study was to determine the expression profile of SHH signaling components and their target genes in ccRCC. Additionally, the present study examined the effects of SHH pathway inhibitory drugs (RU-SKI43, cyclopamine and GLI-antagonist 61) on cell viability, cell cycle progression, expression levels of SHH target genes and migration ability in 786-O, ACHN and HK2 cells. The study also included paired tumor and normal samples from 62 patients with ccRCC. The mRNA levels in clinical samples and cell lines were measured via reverse transcription-quantitative PCR. Cell viability was examined using a sulforhodamine B assay. Flow cytometry was used to investigate cell cycle progression and the migratory rate of cells was assessed using a wound healing assay. High mRNA levels of SHH, smoothened (SMO), glioma-associated zinc finger protein (GLI)1-3, BCL2 apoptosis regulator (BCL2), MYC proto-oncogene (MYC), vascular endothelial growth factor A (VEGFA) and cyclin D1 (CCND1) were observed in the tumor tissues, especially in early ccRCC, according to the TNM stage or World Health Organization/International Society of Urological Pathology (ISUP) grade. High expression levels of VEGFA, as well as low CCND1 mRNA expression, were associated with short overall survival, and increased VEGFA expression was an independent prognostic factor of a poor outcome in patients with advanced ISUP grade (Cox hazard ratio test). Cyclopamine treatment was found to arrest 786-O cells in the G2/M phase and decreased the expression levels of GLI1, BCL2, VEGFA and CCND1. RU-SKI43 inhibited cell migration and decreased the expression levels of BCL2, MYC and CCND1 in ACHN cells. Overall, the results of the present study suggested that SHH signaling may be involved in the early development of ccRCC, and the expression levels of CCND1 and VEGFA may serve as prognostic factors of this disease. Cyclopamine and RU-SKI43 appear to be potential anti-renal cell carcinoma drugs; however, this hypothesis requires verification by further in vivo studies.

Human telomerase reverse transcriptase (hTERT) synergistic with Sp1 upregulate Gli1 expression and increase gastric cancer invasion and metastasis.

Abnormal expression of human telomerase reverse transcriptase (hTERT) has been widely identified in tumors, but the relevant mechanism is not well known. This study aims to investigate the role and mechanism ofhTERTin gastric cancer metastasis. Gastric cancer and adjacent non-tumor tissues were collected and the expression levels of hTERT and Gli1 were detected by immunohistochemistry. The results demonstrated that hTERT and Gli1 expression levels in gastric cancer tissue were significantly higher than adjacent non-tumor tissues. Western blot and quantitative real-time PCR were used to an identified expression of the related protein in BGC-823 and SGC-7901 cells. The interactions betweenhTERTandSp1were tested by co-immunoprecipitation experiments. Chromatin immunoprecipitation was performed to confirm thatSp1andhTERTcould bind to theGli1promoter. Chromatin reimmunoprecipitation assay further demonstrated that bothhTERTandSp1bind to theSp1site of theGli1promoter. Moreover, thehTERT,Sp1, andGli1were upregulate was verified in human gastric cancer tissues. These results showed that the expression levels ofhTERTin GC tissues were strongly closed to the depth of invasion, lymph node metastasis, TNM (tumor, node, metastasis) stage, and distant metastasis. By combiningSp1andGli1promoter,hTERTupregulatedGli1expression and promoted invasion and metastasis of GC cells. Overall, these data provide a new molecular mechanism ofhTERTto promotes gastric cancer progression. Targetingthe hTERT/Sp1/Gli1axis may represent a new therapeutic strategy.

Horizontal transfer of the stemness-related markers EZH2 and GLI1 by neuroblastoma-derived extracellular vesicles in stromal cells

Neuroblastoma (NB) is the most common extracranial pediatric solid cancer originating from undifferentiated neural crest cells. NB cells express EZH2 and GLI1 genes that are known to maintain the undifferentiated phenotype of cancer stem cells (CSC) in NB. Recent studies suggest that tumor-derived extracellular vesicles (EVs) can regulate the transformation of surrounding cells into CSC by transferring tumor-specific molecules they contain. However, the horizontal transfer of EVs molecules in NB remains largely unknown. We report the analysis of NB-derived EVs in bioengineered models of NB that are based on a collagen 1/hyaluronic acid scaffold designed to mimic the native tumor niche. Using these models, we observed an enrichment of GLI1 and EZH2 mRNAs in NB-derived EVs. As a consequence of the uptake of NB-derived EVs, the host cells increased the expression levels of GLI1 and EZH2. These results suggest the alteration of the expression profile of stromal cells through an EV-based mechanism, and point the GLI1 and EZH2 mRNAs in the EV cargo as diagnostic biomarkers in NB.

Heterozygous truncating variants in SUFU cause congenital ocular motor apraxia

PurposeThis study aimed to delineate the genetic basis of congenital ocular motor apraxia (COMA) in patients not otherwise classifiable. MethodsWe compiled clinical and neuroimaging data of individuals from six unrelated families with distinct clinical features of COMA who do not share common diagnostic characteristics of Joubert syndrome or other known genetic conditions associated with COMA. We used exome sequencing to identify pathogenic variants and functional studies in patient-derived fibroblasts. ResultsIn 15 individuals, we detected familial as well as de novo heterozygous truncating causative variants in the Suppressor of Fused (SUFU) gene, a negative regulator of the Hedgehog (HH) signaling pathway. Functional studies showed no differences in cilia occurrence, morphology, or localization of ciliary proteins, such as smoothened. However, analysis of expression of HH signaling target genes detected a significant increase in the general signaling activity in COMA patient–derived fibroblasts compared with control cells. We observed higher basal HH signaling activity resulting in increased basal expression levels of GLI1, GLI2, GLI3, and Patched1. Neuroimaging revealed subtle cerebellar changes, but no full-blown molar tooth sign. ConclusionTaken together, our data imply that the clinical phenotype associated with heterozygous truncating germline variants in SUFU is a forme fruste of Joubert syndrome.

Effect of bevacizumab on expression level of GLI1 and ING4 in colon cancer animal model.

This study aimed to investigate the effect of bevacizumab on GLI1 and ING4 expression in colon cancer animal model. Colon cancer model in rats was induced by azoxymethane (AOM). Bevacizumab was used for the treatment of colon cancer rats. Tumor volume and weight were measured, tumor growth curve was visualized and tumor inhibition rate was calculated. GLI1 and ING4 of colon cancer cells were silencing expressed. Western blot analysis was used to detect the expressions of GLI1, ING4, caspase-3, Bax, β-catenin, Bcl2, PTEN, PI3K, Akt, NF-κB. The apoptosis rate was detected by flow cytometry. MTT assay was used to detect cell activity to get IC50 value. After AOM induced colon cancer model in rats, the expressions of ING4, caspase-3, Bax and PTEN were downregulated, the expressions of GLI1, β-catenin, Bcl2, PI3K, Akt and NF-κB were upregulated and the apoptosis rate was downregulated. After bevacizumab treatment, the tumor volume and weight decreased, the expressions of ING4, caspase-3, Bax, PTEN were upregulated, the expressions of GLI1, β-catenin, Bcl2, PI3K, Akt, NF-κB were downregulated, and the cell apoptosis rate increased. Cell experiments showed that GLI1 promotes tumor growth and reduces the sensitivity of bevacizumab, while ING4 inhibits tumor growth and increases the sensitivity of bevacizumab. Bevacizumab inhibits the growth of colon cancer tumor by upregulating ING4 and downregulating GLI1.

Sonic Hedgehog/Gli1 Signaling Pathway Regulates Cell Migration and Invasion via Induction of Epithelial-to-mesenchymal Transition in Gastric Cancer.

Background: The aberrant activation of the Sonic hedgehog (Shh) signaling pathway is involved in progression of several types of cancer, including gastric cancer (GC). However, it remains uncertain whether it also plays a critical role in promoting cancer initiation and progression by inducing epithelial-to-mesenchymal transition (EMT) in GC. Thus, the aim of the present study was to determine whether the Shh pathway is involved in GC, and to investigate the function of the Shh pathway in the induction of EMT in GC.Materials and methods: The expression levels of Shh pathway members and EMT markers were examined in GC tissues by immunohistochemistry. The association between these factors and patient clinicopathological characteristics was analyzed. In addition, Gli-antagonist 61 (GANT61) was used to block Shh/Gli1 pathway activity, and recombinant Shh proteins (N-Shh) were used to activate the Shh pathway in GC cells. Wound healing and Transwell invasion and migration assays were performed to assess the effects of the Shh pathway on the migration and invasion of GC cells in vitro. Furthermore, western blot analysis was used to examine the changes in protein expression.Results: The results demonstrated that these Shh/Gli1 pathway members were upregulated in GC tissues, and that Gli1 upregulation was associated with tumor progression and a poor prognosis. Gli1 expression was negatively associated with E-cadherin (E-Cad) expression, and positively with Vimentin (VIM) expression in GC specimens. Further analysis revealed that when the Shh/Gli1 pathway was activated, the migratory and invasive abilities of GC cells were enhanced, and the expression levels of Gli1 and VIM were increased, while E-Cad expression was decreased. Opposite results were observed when the Shh/Gli1 pathway was blocked by GANT61.Conclusions: The present study indicated that the Shh/Gli1 pathway exhibits an abnormal activation pattern in GC with possible predictive and prognostic significance. The Shh/Gli1 pathway may promote the migratory and invasive potential of GC cells by inducing EMT. The Shh/Gli1 pathway can thus be considered as a potential therapeutic target for GC.

Regulation and Role of GLI1 in Cutaneous Squamous Cell Carcinoma Pathogenesis.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin tumor in humans. Although current therapies are sufficient to clear the tumor in many cases, the overall risk of cSCC metastasis is still 5%. Alternative treatment options could help to overcome this situation. Here we focused on the role of the Hedgehog (HH) signaling pathway and its interplay with epidermal growth factor receptor (EGFR) signaling in cSCC. The analyses revealed that, despite lack of Sonic HH (SHH) expression, a subset of human cSCC can express GLI1, a marker for active HH signaling, within distinct tumor areas. In contrast, all tumors strongly express EGFR and the hair follicle stem cell marker SOX9 at the highly proliferative tumor-stroma interface, whereas central tumor regions with a more differentiated stratum spinosum cell type lack both EGFR and SOX9 expression. In vitro experiments indicate that activation of EGFR signaling in the human cSCC cell lines SCL-1, MET-1, and MET-4 leads to GLI1 inhibition via the MEK/ERK axis without affecting cellular proliferation. Of note, EGFR activation also inhibits cellular migration of SCL-1 and MET-4 cells. Because proliferation and migration of the cells is also not altered by a GLI1 knockdown, GLI1 is apparently not involved in processes of aggressiveness in established cSCC tumors. In contrast, our data rather suggest a negative correlation between Gli1 expression level and cSCC formation because skin of Ptch +/- mice with slightly elevated Gli1 expression levels is significantly less susceptible to chemically-induced cSCC formation compared to murine wildtype skin. Although not yet formally validated, these data open the possibility that GLI1 (and thus HH signaling) may antagonize cSCC initiation and is not involved in cSCC aggressiveness, at least in a subset of cSCC.

The inhibitory effect and safety of GANT61 on HeLa cells in nude mice

The Hedgehog (Hh) pathway effector Gli1 plays an important role in cervical cancer, and GANT61 is an Hh signaling inhibitor. In this study, we aimed to investigate the inhibitory effect of GANT61 on cervical cancer and to study its safety in nude mice. We used in vivo experiments to assess the effect of GANT61 on the growth of cervical cancer HeLa cells, and we measured the WBC, HGB, PLT, ALT, AST and Cre levels in nude mice. Next, we examined the organ and tumor morphology and distant metastasis by HE staining. We used immunohistochemistry to monitor the expression levels of Gli1, FoxM1, Ki-67, cyclinD1, E-cadherin, vimentin, survivin, caspase-3 and CD34+. Western blotting and RT-RCR were used to measure Gli1 expression. GANT61 inhibited the growth and metastasis of HeLa cervical cancer cells upon their transplantation into nude mice, and we preliminarily propose that GANT61 is safe for nude mice. These findings suggest that GANT61 could be used as a Hedgehog inhibitor to inhibit EMT and proliferation and to promote apoptosis via Gli1 downregulation.

Primary impact of Gli1 on radioresistance in esophageal cancer.

Radioresistance is the primary cause for the low efficacy of radiotherapy in the treatment of esophageal cancer (EC). Increasing evidence has demonstrated that the Sonic Hedgehog (Shh) signaling pathway may be involved in the pathology of various tumors, including EC. The present study aimed to examine the association between radioresistance in EC and the Sonic Hedgehog pathway, and to determine whether a downstream transcription factor of the Shh pathway, glioma-associated oncogene family zinc finger 1 (Gli1), serves a primary role in radioresistance. The radiation-resistant cell line Eca109R was established by repeated low dose (cumulative dose 60 Gy) irradiation of the human EC cell line Eca109. The level of cell radiosensitivity was determined by colony formation assay, and the localization of Gli1 was detected using immunofluorescence. Western blotting was used to determine the protein expression levels of Gli1, Shh, patched 1 (Ptch) and smoothened frizzled class receptor (Smo) in the two cell lines. Significantly higher levels of Gli1 were identified in the Eca109R cell line compared with those inEca109 cells (P<0.05). Additionally, western blotting analysis demonstrated an increased expression level of the Gli1, Shh, Ptch and Smo proteins in Eca109R, compared with Eca109 cells (P<0.05). Overexpression of Gli1 in the parental cell line led to decreased levels of radiosensitivity and radiosensitivity of the radioresistant cell line was restored through knockdown of Gli1. The present study demonstrated that Gli1 may be associated with the development of radioresistance in EC.