Overexpression Of Gli1 Research Articles

The Role of GLI in the Regulation of Hepatic Epithelial-Mesenchymal Transition in Biliary Atresia.

ObjectiveTo study the regulatory role of GLI1/GLI2, a nuclear transcription factor of the Sonic hedgehog (Shh) signaling pathway, in epithelial–mesenchymal transition (EMT) related to hepatic fibrosis in patients with biliary atresia (BA).MethodsThe messenger RNA (mRNA) and protein expression levels of GLI1/GLI2, Snail/Slug, and other Shh- and EMT-related cytokines were tested in the liver tissues of BA patients and animals. Then, GLI1/GLI2 was silenced and overexpressed in mouse intrahepatic bile duct epithelial cells (mIBECs) and BA animals to investigate changes in the mRNA and protein expression of EMT key factors and liver fibrosis indicators. After silencing and overexpression of GLI1/GLI2, immunofluorescence was used to detect the expression of cytokeratin-19 (CK19) and α-smooth muscle actin (α-SMA) in mIBECs, and hematoxylin and eosin (HE) staining and Masson staining were used to observe the degree of liver fibrosis in the BA animals.ResultsCompared with the control, the mRNA and protein expression levels of GLI2, Snail, vimentin, and α-SMA were significantly increased and those of E-cadherin were significantly decreased in liver tissue from BA patients and animals. Overexpression of GLI2 increased the mRNA and protein expression levels of Snail, vimentin, and α-SMA and that of E-cadherin was significantly decreased in mIBECs and BA animals. After GLI2 silencing, the opposite pattern was observed. Immunofluorescence detection showed enhanced expression of the bile duct epithelial cell marker CK19 in mIBECs after GLI2 silencing and enhanced expression of the mesenchymal cell marker α-SMA after GLI2 overexpression. HE and Masson staining suggested that the GLI2-overexpressing group had a significantly higher degree of fibrosis.ConclusionThe Shh signaling pathway plays an important role in fibrogenesis in BA. GLI2 can significantly regulate EMT in mIBECs and livers of BA mice.

Gli1 promotes epithelial-mesenchymal transition and metastasis of non-small cell lung carcinoma by regulating snail transcriptional activity and stability.

Metastasis is crucial for the mortality of non-small cell lung carcinoma (NSCLC) patients. The epithelial–mesenchymal transition (EMT) plays a critical role in regulating tumor metastasis. Glioma-associated oncogene 1 (Gli1) is aberrantly active in a series of tumor tissues. However, the molecular regulatory relationships between Gli1 and NSCLC metastasis have not yet been identified. Herein, we reported Gli1 promoted NSCLC metastasis. High Gli1 expression was associated with poor survival of NSCLC patients. Ectopic expression of Gli1 in low metastatic A549 and NCI-H460 cells enhanced their migration, invasion abilities and facilitated EMT process, whereas knock-down of Gli1 in high metastatic NCI-H1299 and NCI-H1703 cells showed an opposite effect. Notably, Gli1 overexpression accelerated the lung and liver metastasis of NSCLC in the intravenously injected metastasis model. Further research showed that Gli1 positively regulated Snail expression by binding to its promoter and enhancing its protein stability, thereby facilitating the migration, invasion and EMT of NSCLC. In addition, administration of GANT-61, a Gli1 inhibitor, obviously suppressed the metastasis of NSCLC. Collectively, our study reveals that Gli1 is a critical regulator for NSCLC metastasis and suggests that targeting Gli1 is a prospective therapy strategy for metastatic NSCLC.

Saikosaponin B1 and Saikosaponin D inhibit tumor growth in medulloblastoma allograft mice via inhibiting the Hedgehog signaling pathway.

Medulloblastoma (MB), accounting for nearly 10% of all childhood brain tumors, are implicated with aberrant activation of the Hedgehog (Hh) signaling pathway. Saikosaponin B1 (SSB1) and Saikosaponin D (SSD), two bioactive constituents of Radix Bupleuri, are reported to have many biological activities including anticancer activities. In our work, we evaluated the inhibition of SSB1 and SSD on MB tumor growth in allograft mice and explored the underlying mechanisms. The associated biological activity was investigated in Shh Light II cells, an Hh-responsive fibroblast cell line, using the Dual-Glo® Luciferase Assay System. First, SSB1 (IC50, 241.8nM) and SSD (IC50, 168.7nM) inhibited GLI-luciferase activity in Shh Light II cells stimulated with ShhN CM, as well as Gli1 and Ptch1 mRNA expression. In addition, both compounds suppressed the Hh signaling activity provoked by smoothened agonist (SAG) or excessive Smoothened (SMO) expression. Meanwhile, SSB1 and SSD did not inhibit glioma-associated oncogene homolog (GLI) luciferase activity activated by abnormal expression of downstream molecules, suppressor of fuse (SUFU) knockdown or GLI2 overexpression. Consequently, SSB1 (30mg/kg, ip) and SSD (10mg/kg, ip) displayed excellent in vivo inhibitory activity in MB allografts, and the tumor growth inhibition ratios were approximately 50% and 70%, respectively. Our findings, thus, identify SSB1 and SSD significantly inhibit tumor growth in MB models by inhibiting the Hedgehog pathway through targeting SMO.

Hedgehog-Gli2 Signaling Promotes Chemoresistance in Ovarian Cancer Cells by Regulating MDR1.

BackgroundCisplatin (DDP) resistance remains a key challenge in improving the clinical outcome of patients with ovarian cancer (OC). Gli2 overexpression can lead to DDP resistance in OC cells, but the specific underlying regulatory mechanism remains unclear. The membrane transporter encoding gene MDR1 positively regulates chemotherapy resistance in various cancer types. We evaluated MDR1 as a potential Gli2 downstream target and the contribution of the Gli2/MDR1 axis in promoting DDP resistance in OC cells.MethodsTo generate drug-resistant SKOV3/DDP cells, SKOV3 cells were grown for six months under continuous induction wherein the DDP concentration was steadily increased. Gli2 expression in OC cells with varying DDP sensitivities was detected using western blot. Cell counting kit-8 assays were used to assess the DDP sensitivity of SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells and reversal of DDP resistance in SKOV3/DDP and A2780/DDP cells. Cell proliferation was analyzed using 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays. The transcriptional regulation of MDR1 by Gli2 was determined using luciferase reporter assays. Finally, xenograft OC tumors were generated in nude mice, which were then treated with intraperitoneal DDP or phosphate-buffered saline (PBS) injections to investigate if Gli2 affected DDP resistance in OC in vivo.ResultsDDP-resistant SKOV3/DDP and A2780/DDP cells showed higher expression of Gli2 and MDR1 as compared with that in DDP-sensitive OC cells. Gli2 knockdown in SKOV3/DDP cells significantly reduced MDR1 expression, whereas it increased DNA damage, thereby sensitizing OC cells to DDP. Similar results were obtained after targeting Gli2 expression with the Gli-antagonist 61 inhibitor (GANT61) in SKOV3/DDP and A2780/DDP cells. In cells stably overexpressing Gli2, treatment with gradient concentrations of verapamil, an MDR1 inhibitor, significantly inhibited MDR1 expression. Our findings indicate that downregulation of MDR1 expression may reverse OC cell resistance to DDP. Moreover, dual-luciferase reporter gene assays confirmed that MDR1 is a direct downstream target of Gli2, with Gli2 positively regulating MDR1 expression. Finally, subcutaneous xenotransplantation in nude mice demonstrated that Gli2 plays a key role in regulating OC drug resistance.ConclusionsWe identified a mechanism by which Hedgehog-Gli signaling regulates OC chemoresistance by modulating MDR1 expression. Hence, Gli2 and MDR1 are potential biomarkers and therapeutic targets in patients with chemoresistant OC.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone provokes progression from chronic pancreatitis to pancreatic intraepithelial neoplasia

SummaryThe risk of pancreatic cancer is higher among people who are cigarette smokers than among non-smokers; however, the action mechanisms of cigarette metabolites are not yet fully understood. In this study, we investigated the effect of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in cigarette smoking on chronic pancreatitis and pancreatic cancer as well as the biological mechanism of NNK causing malignant transformation. We show that smoking may promote Kras mutation and P16 promoter methylation from clinical samples and NNK markedly facilitates the growth and migration of pancreatic cancer cells via the activation of Sonic Hedgehog signaling. We demonstrate that NNK promotes acinar-to-ductal metastasis and pancreatic intraepithelial neoplasia in rats with chronic pancreatitis, accompanied by desmoplastic reaction and Gli1 overexpression. Together, we here present evidence that NNK provokes the progression of chronic pancreatitis toward pancreatic cancer and highlight potential strategies and targets for early prevention of pancreatic cancer and its therapeutics.

Rosmarinic Acid Decreases the Malignancy of Pancreatic Cancer Through Inhibiting Gli1 Signaling

BackgroundRosmarinic acid (RA) has been shown to exert anti-tumor effects on various types of cancer. However, its roles in the treatment of pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanisms remain elusive. PurposeThe present study aimed to investigate the therapeutic effects of RA on PDAC as well as the underlying mechanisms. Study designEvaluation of the effects of RA on PDAC malignancy both in vitro and in vivo. MethodsCell counting kit 8 (CCK8) assay, colony formation assay, 5-Ethynyl-2′-deoxyuridine (EDU) incorporation assay, cell cycle analysis, and apoptosis assay were conducted to assess the inhibitory effect of RA on PDAC cell proliferation. Meanwhile, western blotting and RT-qPCR assay were performed to detect the target gene expression at protein and mRNA levels, respectively. Moreover, the in vivo anti-tumor activities of RA were assayed in an xenograft mouse model of PDAC. ResultsRA dramatically down-regulated Gli1 and its downstream targets. Further studies showed that RA prevents the nuclear translocation of Gli1, while promoting the degradation of cytosolic Gli1 via the proteasome pathway. Moreover, we observed that RA induced G1/S cell cycle arrest and apoptosis in the PDAC cells through regulating the expression of P21, P27, CDK2, Cyclin E, Bax, and Bcl-2, it inhibited the PDAC cell migration and invasion via E-cadherin and MMP-9. Notably, Gli1 overexpression markedly reversed the above RA-induced effects on PDAC cells, whereas Gli1 knockdown enhanced the effects. Additionally, the in vivo assays demonstrated that RA suppresses the tumor growth of PDAC presumably by inhibiting Gli1. ConclusionWe provided evidence that RA restrained the nuclear translocation of Gli1 and facilitates Gli1 degradation via proteasome pathway, reducing the malignancy of PDAC cells. These findings implicated RA as a therapeutic agent for PDAC.

An Analysis of Structure-function Co-relation between GLI Oncoprotein and HLA Immune-gene Transcriptional Regulation through Molecular Docking

Background:GLI proteins play a significant role in the transduction of the Hedgehog (Hh) signaling pathway. A variety of human cancers, including the brain, gastrointestinal, lung, breast, and prostate cancers, demonstrate inappropriate activation of this pathway. GLI helps in proliferation and has an inhibitory role in the differentiation of hematopoietic stem cells. Malignancies may have a defect in differentiation. Different types of malignancies and undifferentiated cells have a low level of HLA expression on their cell surface.Objective:Human Leukocytic Antigen (HLA) downregulation is frequently observed in cancer cells. This work is aimed to hypothesize whether this downregulation of HLA molecules is GLI oncoprotein mediated or not. To understand the roles of different types of GLI oncoproteins on different classes of HLA transcriptional machinery was carried out through structure-based modeling and molecular docking studies.Methods:To investigate the role of GLI in HLA expression /downregulation is Hh-GLI mediated or not, molecular docking based computational interaction studies were performed between different GLI proteins (GLI1, GLI2, and GLI3) with TATA box binding protein (TBP) and compare the binding efficiencies of different HLA gene (both HLA class I and –II) regulating transcription factors (RelA, RFX5, RFXAP, RFXANK, CIITA, CREB1, and their combinations) with TBP. Due to unavailability of 3D protein structures of GLI2 and cyclin D2 (a natural ligand of GLI1) were modelled followed by structural validation by Ramachandran plot analysis.Results:GLI proteins especially, GLI1 and GLI2, have almost similar binding energy of RFX5-RFXANK- RFXAP and CIITA multi-protein complex to TBP but has lower binding energy between RelA to TBP.Conclusion:This study suggests that HLA class I may not be downregulated by GLI; however, over-expression of GLI1 is may be responsible for HLA class II downregulation. Thus this protein may be responsible for the maintenance of the undifferentiated state of malignant cells. This study also suggests the implicative role of GLI1 in the early definitive stage of hematopoiesis.

Transcriptomic analysis reveals proinflammatory signatures associated with acute myeloid leukemia progression

AbstractNumerous studies have been performed over the last decade to exploit the complexity of genomic and transcriptomic lesions driving the initiation of acute myeloid leukemia (AML). These studies have helped improve risk classification and treatment options. Detailed molecular characterization of longitudinal AML samples is sparse, however; meanwhile, relapse and therapy resistance represent the main challenges in AML care. To this end, we performed transcriptome-wide RNA sequencing of longitudinal diagnosis, relapse, and/or primary resistant samples from 47 adult and 23 pediatric AML patients with known mutational background. Gene expression analysis revealed the association of short event-free survival with overexpression of GLI2 and IL1R1, as well as downregulation of ST18. Moreover, CR1 downregulation and DPEP1 upregulation were associated with AML relapse both in adults and children. Finally, machine learning–based and network-based analysis identified overexpressed CD6 and downregulated INSR as highly copredictive genes depicting important relapse-associated characteristics among adult patients with AML. Our findings highlight the importance of a tumor-promoting inflammatory environment in leukemia progression, as indicated by several of the herein identified differentially expressed genes. Together, this knowledge provides the foundation for novel personalized drug targets and has the potential to maximize the benefit of current treatments to improve cure rates in AML.

Gemcitabine-induced Gli-dependent activation of hedgehog pathway resists to the treatment of urothelial carcinoma cells.

Patients with urothelial carcinoma (UC) experience gemcitabine resistance is a critical issue. The role of hedgehog pathway in the problem was explored. The expressions of phospho-AKTser473, phospho-GSK3βser9 and Gli2 were up-regulated in gemcitabine-resistant NTUB1 (NGR) cells. Without hedgehog ligands, Gli proteins can be phosphorylated by GSK3β kinase to inhibit their downstream regulations. Furthermore, the GSK3β kinase can be phosphorylated by AKT at its Ser9 residue to become an inactive kinase. Therefore, overexpression of AKT1, Flag-GSKS9D (constitutively inactive form) or active Gli2 (GLI2ΔN) in NTUB1 cells could activate Gli2 pathway to enhance migration/invasion ability and increase gemcitabine resistance, respectively. Conversely, overexpression of Flag-GSKS9A (constitutively active form) or knockdown of Gli2 could suppress Gli2 pathway, and then reduce gemcitabine resistance in NGR cells. Therefore, we suggest gemcitabine-activated AKT/GSK3β pathway can elicit Gli2 activity, which leads to enhanced migration/invasion ability and resistance to gemcitabine therapy in UC patients. The non-canonical hedgehog pathway should be evaluated in the therapy to benefit UC patients.

Smoothened loss is a characteristic of neuroendocrine prostate cancer.

PurposeHedgehog (Hh) signaling promotes castration‐resistant prostate cancer by supporting androgen‐independent prostate cancer cell development and growth; however, its role in neuroendocrine prostate cancer (NEPC) has not yet been explored. In this study, we assessed the expression of key genes involved in Hh signaling in prostate cancer and investigated the potential role of smoothened (SMO) in the pathogenesis of NEPC.MethodsSix public datasets, each containing cases of prostate adenocarcinoma (AdPC) and NEPC, were analyzed to compare the differential messenger RNA (mRNA) expression of six classic Hh signaling genes. The SMO, synaptophysin, chromogranin A (CHGA) and androgen receptor (AR) proteins were evaluated in human tissues from 5 cases of NEPC, 2 cases of AdPC mixed with NEPC, 2 cases of AdPC with neuroendocrine differentiation and 22 cases of high‐grade AdPC as determined by an immunohistochemistry assay. Gene set enrichment analysis (GSEA) was performed to identify relevant genetic signatures associated with SMO expression based on the public datasets. Stable SMO‐knockdown LNCaP and C4‐2B cells were established with a lentiviral system, and the expression of SMO, Gli1, AR, prostate‐specific antigen (PSA), and REST was assessed by real‐time polymerase chain reaction and western blot. Secreted PSA in the conditioned medium was assessed by ELISA. Gli1 was ectopically expressed performed by the transfection of Gli1 complementary DNA into SMO‐knockdown LNCaP cells, and western blot was used to assess of AR and PSA expression.ResultsThe mRNA level of SMO was dramatically downregulated in NEPC samples compared with AdPC samples in all 6 public datasets. SMO protein loss was observed in 100% of NEPC samples but in only 9% (2 of 22) of high‐grade AdPC samples. GSEA results showed that SMO loss was closely correlated with AR signaling activity. Stable SMO knockdown significantly attenuated AR signaling activity and suppressed AR expression, while Gli1 overexpression partially reversed the inhibitory effects of SMO knockdown on AR signaling activity and AR expression in LNCaP and C4‐2B cells.ConclusionThese results demonstrate that SMO loss is a characteristic of NEPC and that detecting SMO by IHC could aid pathologists in NEPC diagnosis. SMO loss may promote NEPC pathogenesis by modulating AR signaling.

Inhibition of the sonic hedgehog pathway activates TGF-\u03b2-activated kinase (TAK1) to induce autophagy and suppress apoptosis in thyroid tumor cells

The sonic hedgehog (Shh) pathway is highly activated in a variety of malignancies and plays important roles in tumorigenesis, tumor growth, drug resistance, and metastasis. Our recent study showed that the inhibitors of the Shh pathway such as cyclopamine (CP), a Smothened (SMO) inhibitor, and GANT61, a Gli1 inhibitor, have modest inhibitory effects on thyroid tumor cell proliferation and tumor growth. The objective of this study was to determine whether autophagy was induced by inhibition of the Shh pathway and could negatively regulate GANT61-induced apoptosis. Here we report that inhibition of the Shh pathway by Gli1 siRNA or by cyclopamine and GANT61 induced autophagy in SW1736 and KAT-18 cells, two anaplastic thyroid cancer cell lines; whereas Gli1 overexpression suppressed autophagy. Mechanistic investigation revealed that inhibition of the Shh pathway activated TAK1 and its two downstream kinases, the c-Jun-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). GANT61-induced autophagy was blocked by TAK1 siRNA and the inhibitors of TAK1 (5Z-7-oxozeaenol, 5Z), JNK (SP600125), and AMPK (Compound C, CC). Inhibition of autophagy by chloroquine and 5Z and by TAK1 and Beclin-1 siRNA enhanced GANT61-induced apoptosis and its antiproliferative activity. Our study has shown that inhibition of the Shh pathway induces autophagy by activating TAK1, whereas autophagy in turn suppresses GANT61-induced apoptosis. We have uncovered a previously unrecognized role of TAK1 in Shh pathway inhibition-induced autophagy and apoptosis.

Constitutive GLI1 expression in chondrosarcoma is regulated by major vault protein via mTOR/S6K1 signaling cascade

Hedgehog signaling plays a pivotal role in embryonic pattern formation and diverse aspects of the postnatal biological process. Perturbation of the hedgehog pathway and overexpression of GLI1, a downstream transcription factor in the hedgehog pathway, are highly relevant to several malignancies including chondrosarcoma (CS). We previously found that knocking down expression of GLI1 attenuates the disrupted Indian hedgehog (IHH) signal pathway and suppresses cell survival in human CS cells. However, the underlying mechanisms regulating the expression of GLI1 are still unknown. Here, we demonstrated the implication of GLI1 in SMO-independent pathways in CS cells. A GLI1 binding protein, major vault protein (MVP), was identified using the affinity purification method. MVP promoted the nuclear transport and stabilization of GLI1 by compromising the binding affinity of GLI1 with suppressor of fused homolog (SUFU) and increased GLI1 expression via mTOR/S6K1 signaling cascade. Functionally, knockdown of MVP suppressed cell growth and induced apoptosis. Simultaneous inhibition of MVP and GLI1 strongly inhibits the growth of CS in vitro and in vivo. Moreover, IHC results showed that MVP, GLI1, and P-p70S6K1 were highly expressed and positively correlated with each other in 71 human CS tissues. Overall, our findings revealed a novel regulating mechanism for HH-independent GLI1 expression and provide a rationale for combination therapy in patients with advanced CS.

MiR-636 inhibits EMT, cell proliferation and cell cycle of ovarian cancer by directly targeting transcription factor Gli2 involved in Hedgehog pathway

BackgroundHedgehog (Hh) signaling pathway, which is essential for cell proliferation and differentiation, is noted to be aberrantly activated in tumor from increasing studies in recent years. MicroRNAs (miRNAs) as an important non-coding RNA in cells have been proven to possess a regulatory role specific to the Hh signaling pathway. Here, in vitro and in vivo cellular/molecular experiments were adopted to clarify the regulatory mechanism linking miR-636 to the Hh signaling pathway in ovarian cancer (OVC).MethodsProtein–protein interaction analysis was performed to identify the hub gene in the Hh pathway. TargetScan database was used to predict the potential upstream regulators for Gli2. qRT-PCR was performed to test the expression of miR-636, while Western blot was conducted to detect the expression of proteins related to the Hh pathway and epithelial-mesenchymal transition (EMT). For cell functional experiments, HO-8910PM OVC cell line was used. MTT assay and wound healing assay were used to measure the effect of miR-636 on cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used to identify the change in expression of Hh and EMT-related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeting relationship between miR-636 and Gli2. Xenotransplantation models were established for in vivo examination.ResultsGli2 was identified as the hub gene of the Hh pathway and it was validated to be regulated by miR-636 based on the data from TargetScan and GEO databases. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines, and overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation, migration and induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 caused opposite results. Dual-luciferase reporter gene assay revealed that Gli2 was the target gene of miR-636 in OVC. Besides, overexpressed miR-636 decreased protein expression of Gli2, and affected the expression of proteins related to the Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration, and attenuated the blocking effect of miR-636 on cell cycle. The xenotransplantation experiment suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process of OVC cells via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation in vivo.ConclusionmiR-636 mediates the activation of the Hh pathway via binding to Gli2, thus inhibiting EMT, suppressing cell proliferation and migration of OVC.Trial registration: The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine (IR2019001235). Written informed consent was obtained from individual or guardian participants.

WISP-1 Induced by Mechanical Stress Contributes to the Fibrosis and Hypertrophy of Ligamentum Flavum Through the Hedgehog-Gli1 Signaling

Background: The chronic fibrosis and hypertrophy of ligamentum flavum (LF) is an important cause of lumbar spinal canal stenosis (LSCS). Our previous work showed WNT1 inducible signaling pathway protein 1 (WISP-1) is a critical driver of LF fibrosis. However, its potential mechanism has not been explored. Methods: We studied the expression of Hedgehog-related proteins in human LF tissues. Cell viability, cell cycle, apoptosis rate and molecular mechanisms were evaluated in LF cells with lentivirus-mediated knockdown or over-expression of WISP-1 and Gli1. Finally, the molecular mechanism was confirmed in in vivo studies. Findings: The results showed Gli1 was upregulated in hypertrophic LF tissues and required for fibroblast proliferation, apoptosis inhibition and collagen expression in fibroblasts. Moreover, mechanical stretching increased the expression of WISP-1 in LF fibroblasts. Furthermore, WISP-1 induced fibrogenesis in vitro through Hedgehog-Gli1 pathway. This conclusion was supported by the fact that WISP-1 activated Hedgehog-Gli1 pathway in LF fibroblasts and cyclopamine attenuated the effect of WISP-1-induced fibrogenesis. WISP-1 also promoted the transition of fibroblasts into myofibroblasts via Hedgehog pathway. Importantly, hypertrophic LF rabbit model induced by mechanical stress also showed pathological change of fibrosis and more higher expression of WISP-1, Gli1 and ɑ-SMA. Therapeutic administration of cyclopamine reduced collagen expression, fibroblast proliferation, myofibroblast differentiation and ameliorated fibrosis in rabbit model of mechanical stress-induced LF fibrosis. Interpretation: Our study showed the mechanical stress/WISP-1/Hedgehog signaling as a new fibrotic axis contributing to the LF hypertrophy and identified Hedgehog signaling as a therapeutic target for the prevention and treatment of LF fibrosis. Funding Statement: This work was supported by the National Natural Science Foundation of China (Grant No. 81802198) and Natural Science Foundation of Jiangsu Province (Grant No. SBK2019022658). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: An informed consent form was signed by the all patients and the study was approved by the Ethics Committee of Nanjing Medical University (Approval No.20180104).

Clinical value and potential association of Rab1A and FoxM1 aberrant expression in colorectal cancer

Colorectal carcinoma (CRC) is one of the most common malignancies with a dismal 5-year survival rate. Our recent study indicated that Rab1A expression was closely related to GLI1 expression. A previous study shows that aberrant overexpression of GLI1 promotes colorectal cancer metastasis via FoxM1 overexpression. However, the potential correlation between Rab1A and FoxM1 in CRC remains elusive. Immunohistochemistry was performed to investigate the association of the expression of Rab1A and FoxM1 and to determine the prognosis in 135 CRC tissue and adjacent normal tissues. Using Oncomine datasets, we found that Rab1A and FoxM1 mRNA were obviously upregulated in CRC tissues compared to normal tissues. Additionally, the expression of Rab1A and FoxM1 was significantly higher in CRC tissues than that in normal tissues. Rab1A expression was positively correlated with FoxM1 expression in CRC, especially in TNM stage III. In addition, Rab1A and FoxM1 overexpression was found to be significantly correlated with poor prognosis in CRC patients. Besides, both high expression of Rab1A and FoxM1 led to a worse prognosis than anyone low group, and both low expression of Rab1A and FoxM1 had a better prognosis than the anyone low group. Therefore, Rab1A and FoxM1 play crucial roles and could be used as clinical biomarkers in CRC.

Genomic and Transcriptomic Characterization of Adult and Pediatric Relapsed Acute Myeloid Leukemia Reveals Novel Therapeutic Targets

Acute myeloid leukemia (AML) is the overall most common form of acute leukemia, characterized by a high relapse frequency and low long-term survival rate especially for adults. Even though children with AML have a better prognosis than adults, patients with recurrent AML - independent of age - show poorer overall survival, accelerated disease progression and they often do not respond to conventional treatment. Therefore, a more personalized treatment approach is required to prolong event free survival (EFS) and expand treatment options for relapse and primary resistant (R/PR) AML patients. During the last decade, AML research has largely been focused on improving diagnostic and prognostic tools in AML by investigating data derived from whole genome- and whole exome sequencing (WGS and WES, respectively), gene panels or differential expression analyses of individual genes and gene-fusions. Nevertheless, studies on relapsing AML incorporating WGS data are rare, although necessary to investigate the full repertoire of genetic aberrations underlying AML progression and therapy resistance. To this end, we applied a combination of WGS (WES for a subset of cases) and RNA-seq of longitudinal samples from 48 adult and 21 pediatric R/PR AML cases from the Nordic countries. These comprised tumor samples collected at diagnosis (n=49) and relapse (n=76), as well as PR specimens (n=6). Normal bone marrow (BM) derived stromal cells were cultivated from leukemic BM as a source of patient-matched constitutional DNA, while CD34+ BM cells from five healthy donors were used as a source of normal control RNA. Our findings reveal recurrent relapse specific mutations in CSF1R (2.9% of relapse cases) not previously reported in de novo AML, suggesting the use of receptor tyrosine kinase inhibitors as a novel therapeutic option for a subset of AML relapse cases. Further, we report specific differences in the mutational spectrum between adult and pediatric R/PR AML. In adults, we detected higher mutational frequencies of, for instance, ARID1A (6.3%), H3F3A (6.3%) and MGA (10.4%) compared with previous AML studies of only specimens from initial diagnosis, while these mutations were not seen in pediatric AML. In contrast, internal tandem duplications (ITDs) in UBTF were detected solely in pediatric relapsing AML (n=3 [14.3%]). IKZF1 was more frequently mutated in pediatric R/PR AML (14.3%) than previously reported (0.5-2.7%; Bolouri et al., Nat Med. 2018; Shiba et al., Br J Haematol, 2016). Also, differential gene expression analysis identified IKZF1 as downregulated in pediatric chemotherapy resistant samples in comparison with treatment responsive counterparts, independent of IKZF1 mutational status. By investigating differential gene expression patterns of longitudinal samples, we found lower expression of the complement inhibitor CR1/CD35 at relapse compared to their patient matched diagnostic samples in both adults and children. Additionally, IL1R1, encoding a key regulator of inflammation and immune response, was upregulated in both adult and pediatric diagnosis specimens from cases with short EFS, indicating a pronounced role of chronic inflammation during disease progression and AML cell survival. Finally, our findings reveal overexpression of GLI2 and SGMS2 among samples associated with short EFS. Overexpression of these genes may prevent excessive cell proliferation while increasing stemness and dormancy, leading to increased chemotherapy resistance and shorter EFS. Taken together, our results emphasize the advantage of applying a combination of WGS and RNA-seq, to be able to gain a more complete picture of alterations, including mutations, gene fusions and copy number alterations combined with gene expression analysis, when attempting to characterize AML at relapse. This is the first study of both adult and pediatric AML incorporating WGS and RNA-seq analyses on sequential AML samples. Knowledge gathered from this study has provided critical new insights into the biologic basis of this complex disease and will hopefully help to pave the way for improved and individualized treatment strategies. Disclosures No relevant conflicts of interest to declare.

The Hedgehog signaling pathway promotes chemotherapy resistance via multidrug resistance protein1 in ovarian cancer.

Various studies have revealed that the Hedgehog (Hh) signaling pathway promotes ovarian cancer invasion, migration and drug resistance. Previous studies by our group have identified a set of genes, including multidrug resistance gene 1 (MDR1), that are regulated by Hh signaling in ovarian cancer. However, the association between Hh signaling activation and MDR1 expression requires further validation. In the present study, reverse transcription-quantitative PCR or western blot assays were used to evaluate the mRNA and protein expression levels of MDR1, Sonic Hh (Shh), glioma-associated oncogene 2 (Gli2), Gli1 and γ-phosphorylated H2A.X variant histone (γ-H2AX). MTT and colony-formation assays were performed to determine the effect of cisplatin (DDP) after inhibiting the Hh pathway in ovarian cancer cells. The results indicated that MDR1, Gli2 and Shh levels were much higher in SK-OV-3 cells with acquired DDP resistance than in native SK-OV-3 cells. ES-2 cells with overexpression of Gli2 were capable of efficiently forming colonies in the presence of low DDP concentrations. By contrast, Gli2 knockdown in SK-OV-3 cells decreased the colony-forming ability under the same concentration of DDP. As determined by MTT assays, knockdown of Gli2 or targeting of the Hh signaling pathway with either Gli-antagonist 61 (GANT61) or cyclopamine, in combination with DDP treatment, diminished the viability of ES-2 and SK-OV-3 cells, whereas Gli2 overexpression increased the viability of ES-2 cells in the presence of DDP. Knockdown of Gli2 or targeting the Hh signaling pathway with GANT61 also increased γ-H2AX levels but decreased the expression of MDR1 in the presence of DDP. MDR1 expression is regulated by the Hh signaling pathway and is likely a downstream transcription factor of Gli2. In conclusion, targeting the Hh signaling pathway increases the sensitivity of ovarian cancer to DDP. MDR1 is a target gene of the Hh signaling pathway and this pathway may affect chemoresistance of ovarian cancer to DDP via MDR1.

Impaired Hedgehog-Gli1 Pathway Activity Underlies the Vascular Phenotype of Polycystic Kidney Disease.

Polycystic kidney disease (PKD) has been linked to abnormal structure/function of ciliary proteins, leading to renal dysfunction. Recently, attention has been focused in the significant vascular abnormalities associated with PKD, but the mechanisms underlying this phenomenon remain elusive. Here, we seek to define the molecular events regulating the angiogenic imbalance observed in PKD. Using micro computed tomography (n=7) and protein expression analysis (n=5), we assessed the vascular density and the angiogenic profile of noncystic organs in a well-established PKD rat model (Polycystic Kidney-PCK rat). Heart and lungs of PCK rats have reduced vascular density and decreased expression of angiogenic factors compared with wild type. Similarly, PCK-vascular smooth muscle cells (VSMCs; n=4) exhibited lower levels of vascular markers. Then, using small interfering RNA (n=4), we determined the role of the ciliary protein fibrocystin in wild type-VSMCs, a critical component/regulator of vascular structure and function. Reduction of fibrocystin in wild type-VSMCs (n=4) led to an abnormal angiogenic potential similar to that observed in PCK-VSMCs. Furthermore, we investigated the involvement of the hedgehog signaling, a pathway closely linked to the primary cilium and associated with vascular development, in PKD. Mechanistically, we demonstrated that impairment of the hedgehog signaling mediates, in part, this abnormal angiogenic phenotype. Lastly, overexpression of Gli1 in PCK-VSMCs (n=4) restored the expression levels of proangiogenic molecules. Our data support a critical role of fibrocystin in the abnormal vascular phenotype of PKD and indicate that a dysregulation of hedgehog may be responsible, at least in part, for these vascular deficiencies.

Transcriptomic and Proteomic Analysis of Steatohepatitic Hepatocellular Carcinoma Reveals Novel Distinct Biologic Features.

Steatohepatitic hepatocellular carcinoma is a distinct variant of hepatocellular carcinoma strongly associated with underlying nonalcoholic steatohepatitis. The molecular biology of steatohepatitic hepatocellular carcinoma is not fully elucidated, and thus we aimed to investigate the molecular underpinnings of this entity. Transcriptomic analysis using RNAseq was performed on eight tumor-nonneoplastic pairs of steatohepatitic hepatocellular carcinoma with comparison to conventional hepatocellular carcinoma transcriptomes curated in The Cancer Genome Atlas. Immunohistochemistry was used to validate key RNA-level findings. Steatohepatitic hepatocellular carcinoma demonstrated a distinctive differential gene expression profile compared with The Cancer Genome Atlas curated conventional hepatocellular carcinomas (n = 360 cases), indicating the distinctive steatohepatitic hepatocellular carcinoma morphology is associated with a unique gene expression profile. Pathway analysis comparing tumor-nonneoplastic pairs revealed significant upregulation of the hedgehog pathway based on GLI1 overexpression and significant downregulation of carnitine palmitoyltransferase 2 transcript. Glutamine synthetase transcript was significantly upregulated, and fatty acid binding protein 1 transcript was significantly downregulated and immunohistochemically confirmed, indicating steatohepatitic hepatocellular carcinoma tumor cells display a zone 3 phenotype. Steatohepatitic hepatocellular carcinoma demonstrates a distinctive morphology and gene expression profile, phenotype of zone 3 hepatocytes, and activation of the hedgehog pathway and repression of carnitine palmitoyltransferase 2, which may be important in tumorigenesis.

Sodium Butyrate Combined with Docetaxel for the Treatment of Lung Adenocarcinoma A549 Cells by Targeting Gli1.

PurposeThis study is aimed to investigate the combined treating efficacy of sodium butyrate and docetaxel on proliferation and apoptosis of the lung adenocarcinoma A549 cell line based on Gli1 regulation in vitro and in vivo.Materials and MethodsRNA interference method was used to overexpress Gli1 in A549 cells. Cells were treated with varying concentrations of sodium butyrate, docetaxel or both in combination. CCK-8, colony formation assay, Hoechst 33258 staining, flow cytometry and TUNEL assay were employed to detect proliferation, cell cycle and apoptosis. qRT-PCR and Western blot analysis were applied to detect the mRNA and protein expression of Gli1. In vivo tumorigenicity was detected by tumor transplantation in nude mice. Downstream protein levels of Gli1 were detected using Western blot assay.ResultsIt was found that sodium butyrate or docetaxel alone, respectively, inhibited proliferation and promoted apoptosis of A549 cells in vitro and in vivo, while the combination of the two generated significantly higher responses, which were also effective in another lung adenocarcinoma cell line H1299. Furthermore, the combined therapy had an additive effect in suppressing Gli1 expression and regulating the expression of its downstream proteins that involve in proliferation, cell cycle and apoptosis of A549 cells in vitro and in vivo, including decreased protein expression of Ki-67, CDK1, CDK2, Cyclin D1, Bcl-2 and Survivin, and increased protein expression of Cyclin A, p21, Bax and cleaved-Caspase 3. On the other hand, Gli1 overexpression perceptibly reversed the above-mentioned additive effect in vitro and in vivo.ConclusionThis study demonstrates that the combined therapy of sodium butyrate and docetaxel additively inhibits proliferation and promotes apoptosis of A549 lung adenocarcinoma cells via suppressing Gli1 expression in vitro and in vivo. Targeting Gli1 by the combined therapy may provide new insights into the therapeutic management of patients with lung adenocarcinoma.