Hh Signaling Pathway Research Articles

Responsiveness to Hedgehog Pathway Inhibitors in T-Cell Acute Lymphoblastic Leukemia Cells Is Highly Dependent on 5'AMP-Activated Kinase Inactivation.

Numerous studies have shown that hedgehog inhibitors (iHHs) only partially block the growth of tumor cells, especially in vivo. Leukemia often expands in a nutrient-depleted environment (bone marrow and thymus). In order to identify putative signaling pathways implicated in the adaptive response to metabolically adverse conditions, we executed quantitative phospho-proteomics in T-cell acute lymphoblastic leukemia (T-ALL) cells subjected to nutrient-depleted conditions (serum starvation). We found important modulations of peptides phosphorylated by critical signaling pathways including casein kinase, mammalian target of rapamycin, and 5′AMP-activated kinase (AMPK). Surprisingly, in T-ALL cells, AMPK signaling was the most consistently downregulated pathway under serum-depleted conditions, and this coincided with increased GLI1 expression and sensitivity to iHHs, especially the GLI1/2 inhibitor GANT-61. Increased sensitivity to GANT-61 was also found following genetic inactivation of the catalytic subunit of AMPK (AMPKα1) or pharmacological inhibition of AMPK by Compound C. Additionally, patient-derived xenografts showing high GLI1 expression lacked activated AMPK, suggesting an important role for this signaling pathway in regulating GLI1 protein levels. Further, joint targeting of HH and AMPK signaling pathways in T-ALL cells by GANT-61 and Compound C significantly increased the therapeutic response. Our results suggest that metabolic adaptation that occurs under nutrient starvation in T-ALL cells increases responsiveness to HH pathway inhibitors through an AMPK-dependent mechanism and that joint therapeutic targeting of AMPK signaling and HH signaling could represent a valid therapeutic strategy in rapidly expanding tumors where nutrient availability becomes limiting.

Constitutive Hedgehog/GLI2 signaling drives extracutaneous basaloid squamous cell carcinoma development and bone remodeling.

Uncontrolled activation of the Hedgehog (Hh) signaling pathway, operating through GLI transcription factors, plays a central role in the pathogenesis of cutaneous basal cell carcinoma and contributes to the development of several malignancies arising in extracutaneous sites. We now report that K5-tTA;tetO-Gli2 bitransgenic mice develop distinctive epithelial tumors within their jaws. These tumors consist of large masses of highly proliferative, monomorphous, basaloid cells with scattered foci of keratinization and central necrosis, mimicking human basaloid squamous cell carcinoma (BSCC), an aggressive upper aerodigestive tract tumor. Like human BSCC, these tumors express epidermal basal keratins and differentiation-specific keratins within squamous foci. Mouse BSCCs express high levels of Gli2 and Hh target genes, including Gli1 and Ptch1, which we show are also upregulated in a subset of human BSCCs. Mouse BSCCs appear to arise from distinct epithelial sites, including the gingival junctional epithelium and epithelial rests of Malassez, a proposed stem cell compartment. Although Gli2 transgene expression is restricted to epithelial cells, we also detect striking alterations in bone adjacent to BSCCs, with activated osteoblasts, osteoclasts and osteal macrophages, indicative of active bone remodeling. Gli2 transgene inactivation resulted in rapid BSCC regression and reversal of the bone remodeling phenotype. This first-reported mouse model of BSCC supports the concept that uncontrolled Hh signaling plays a central role in the pathogenesis of a subset of human BSCCs, points to Hh/GLI2 signaling as a potential therapeutic target and provides a powerful new tool for probing the mechanistic underpinnings of tumor-associated bone remodeling.

Effectiveness, safety and utilization of vismodegib in locally advanced basal cell carcinoma under real-world conditions in Germany - The non-interventional study NIELS.

Basal cell carcinoma (BCC) can arise by the uncontrolled proliferation of cells from multiple epidermal compartments due to aberrant activation of the Hedgehog (Hh) signalling pathway. Vismodegib, a small-molecule inhibitor of this pathway, is approved for treatment of patients with locally advanced (la) BCC inappropriate for surgery or radiotherapy or patients with symptomatic metastatic (m) BCC. The aim of this non-interventional study was to assess effectiveness with a special focus on duration of response (DOR), safety and utilization of vismodegib for treatment of laBCC in daily practice in Germany. This non-interventional study (NIS) observed treatment of laBCC with vismodegib according to the German label in clinical practice. All available patients who had received at least one dose of vismodegib between commercial availability of vismodegib in Germany (02 August 2013) and 3 years before end of study (31 March 2016) could be included and were documented retrospectively and/or prospectively for up to 3 years. Primary effectiveness variable was DOR. Assessment of tumour response was carried out by the treating physicians. Exploratory variables included utilization of vismodegib, decision makers for therapy and method of tumour response evaluation. All statistical analyses were descriptive. Between September 2015 and March 2019, 66 patients were observed at 26 German centres. The objective response rate (ORR) was 74.2% and the disease control rate (DCR) was 90.9%. The median DOR was 15.9 months (95% CI: 9.2; 25.7; n=49 patients with response). The median progression-free survival (PFS) was 19.1 months and the median time to response (TTR) 2.7 months. A total of 340 adverse events were reported in 63 (95.5%) patients; no new safety signals were identified. The NIS NIELS shows effectiveness and safety of vismodegib in patients with laBCC. It confirms the transferability of the results of the pivotal trial into routine clinical practice.

Positive Crosstalk Between Hedgehog and NF-κB Pathways Is Dependent on KRAS Mutation in Pancreatic Ductal Adenocarcinoma.

It has been shown that aberrant activation of the Hedgehog (Hh) and nuclear factor-kappa B (NF-κB) signaling pathways plays an important role in the pancreatic carcinogenesis, and KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDAC). Until now, the role of KRAS mutation in the context of crosstalk between Hh and NF-κB signaling pathways in PDAC has not been investigated. This study was to determine whether the crosstalk between the Hh and NF-κB pathways is dependent on KRAS mutation in PDAC. The correlation between Gli1, Shh, NF-κB p65 expression and KRAS mutation in PDAC tissues was firstly examined by immunohistochemistry. Next, Western blotting, qPCR, and immunofluorescence were conducted to examine the biological effects of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) as NF-κB signaling agonists, Shh as an Hh ligand alone or in combination with KRAS small interfering RNA (si-KRAS) in KRAS-mutant PDAC cells (MT-KRAS; SW1990 and Panc-1), wild-type KRAS PDAC cells (WT-KRAS; BxPC-3) and mutant KRAS knock-in BxPC-3 cells in vitro as well as tumor growth in vivo. KRAS mutation-dependent crosstalk between Hh and NF-κB in PDAC cells was further assessed by Ras activity and luciferase reporter assays. The aberrant Hh and NF-κB pathway activation was found in PDAC tissues with KRAS mutation. The same findings were confirmed in MT-KRAS PDAC cells and MT-KRAS knock-in BxPC-3 cells, whereas this activation was not observed in WT-KRAS PDAC cells. However, the activation was significantly down-regulated by KRAS silencing in MT-KRAS PDAC cells. Furthermore, MT-KRAS cancer cell proliferation and survival in vitro and tumor growth after inoculation with MT-KRAS cells in vivo were promoted by NF-κB and Hh signaling activation. The pivotal factor for co-activation of NF-κB and Hh signaling is MT-KRAS protein upregulation, showing that positive crosstalk between Hh and NF-κB pathways is dependent upon KRAS mutation in PDAC.

In Vivo Effects of Osteoblast-Specific Overexpression of Gα s and Gα q/11 on Bone Formation and Bone Remodeling During Fracture Healing

Although the regenerative potential of bone is high, fracture healing is sometimes compromised resulting in fracture non-union. Successful prevention and treatment of non-union rely on accurate prognosis of fracture and treatment assignment but are often impeded by a high degree of heterogeneity in fracture pathogenesis and treatment response. Burgeoning evidence that indicates differential levels of G proteins among healthy populations offers a plausible explanation, since G protein signaling plays regulatory roles in bone development and remodeling. We previously demonstrated the in vivo effects of G protein level variation on bone development by characterizing the skeletal phenotypes of two transgenic mouse lines that overexpress normal Gα s (Gs-Tg) or Gα 11 (G11-Tg) in osteoblast lineage cells under the control of the 3.6-kb Col1a1 promoter. Gs-Tg mice showed high bone mass but diminished bone quality due to increased formation of woven bone and cortical porosity. Conversely, G11-Tg mice displayed a low bone mass phenotype with reduced bone strength primarily due to increased bone resorption. It still remains unclear, however, how variation in G protein signaling in osteoblasts affects the rate and quality of bone repair. To characterize bone healing in Gs-Tg and G11-Tg mice, we induced a stabilized transverse osteotomy in the tibia of wild-type, Gs-Tg or G11-Tg at 8 weeks of age and assessed the progression of bone healing at the tissue and molecular levels by Micro-CT, histomorphometry, and quantitative gene expression analysis at 1, 2, 3, and 4 weeks post-fracture. The effects of increased G protein signaling during repair on Wnt and Hh signaling, the two signaling pathways essential in osteogenesis, were also examined by assessing mRNA levels of key components of each pathway. Persistent upregulation of Wnt and Hh signaling in Gs-Tg fractures led to enhanced callus mineralization and new woven bone formation at the expense of cartilaginous callus formation and defective bone remodeling. G11-Tg fractures, in contrast, showed minimal changes in Wnt and Hh pathways and consequently led to only subtle changes in the healing response with a transient increase in callus mineralization followed by its rapid resorption. Our results indicate that G protein signaling in osteoblasts regulates bone formation and remodeling during fracture repair in part by tightly controlling the strength and duration of Wnt and Hh signaling pathways.

Hedgehog signaling interfaces with the Hexoseamine Biosynthetic Pathway to promote M2 polarization of mammary tumor associated macrophages

Abstract The Hedgehog (Hh) signaling pathway is aberrantly activated in breast cancer, and contributes to tumor cell survival, proliferation, and migration. The success of these events is heavily influenced by the surrounding immune microenvironment. One such cell type, macrophages (MΦ) can make up to 50% of the tumor mass. MΦs can be polarized to M1 (tumor-eradicating) or M2 (tumor-promoting), and a high M2/M1 ratio within the tumor microenvironment (TME) is correlated with worse prognosis. We have uncovered a novel role for Hh signaling in promoting M2 polarization. We propose that the mechanism underlying this phenomenon is due to changes in the Hexoseamine Biosynthetic Pathway and consequently cellular O-GlcNAcylation. M2 MΦs have considerably higher levels of UDP-GlcNAc, the substrate for O-GlcNAcylation, compared to M1s, but the consequences of this are unknown. We hypothesize that O-GlcNAc modifications on key M2-specific proteins contribute to enhanced M2 phenotype and function within the TME, and that this phenomenon can be reversed with Hh blockade. Methodology: mouse model of mammary carcinoma, flow cytometry, RNA-seq, metabolomics, IP. This study is the first to elucidate Hh signaling’s role in recalibrating the metabolism of tumor-supporting MΦs. Inhibition of Hh signaling reduces MΦ O-GlcNAcylation resulting in weakened suppressive properties of M2 MΦs through the reduced activity of STAT6. Moreover, mitigating O-GlcNAcylation through Hh blockade pivots M2 MΦs to resemble M1s. Our work provides evidence for modulating Hh activity, and consequently MΦ metabolism, to sculpt the plasticity of MΦs. This knowledge will help us to better understand how to target and diminish the pro-tumorigenic phenotypes of MΦs in the TME.

Investigating the role of Wnt and related signaling pathways in breast cancer patients: Thesis Abstract

Thesis Abstract Background: Breast cancer is a heterogeneous malignant tumor that includes several types of cells. Among them, only breast cancer stem cells (BCSCs) have tumorigenic ability. In this study we tested the hypothesis that BCSCs are behind disparities in behavior and biology of breast cancer between different ethnic groups and it is very important to investigate signaling pathways regulating BCSCs in each population. Therefore, we investigated the role of WNT, HH and apoptosis signaling pathways together with ALDH1A1 and CD44 in BCSCs resistance to chemotherapy in tumor biopsies collected from Egyptian patients compared to British patients. Aim: We investigated the expression levels of specific members of Wnt, Hedgehog, and apoptosis signaling pathways in BCSCs isolated from treated Egyptian and British patients. The change in their expression levels between the two populations was detected. Materials and Methods: Breast Cancer tumors were collected from Egyptian and British patients, dissociated to a single cell suspension in collagenase IV containing buffer. CSCs were isolated using anoikis resistance assay from treated and untreated samples. RNA was extracted and cDNA was synthesized. Custom qPCR arrays were used to detect the gene expression of Wnt5A, CXCR4, LEF1, TCF4, SHH, GLI1, BCL2, XIAP, ALDH1A1and CD44. Data were analyzed by ΔΔCt method. Results: Our data showed a number of genes with different expression levels in the treated Egyptian patients compared to the treated British patients. Conclusion: Molecular mechanisms that regulate BCSCs differ from a population to another. Moreover, the different treatment regimens in different countries may affect the way BCSCs resist therapy.

Fused: a promising molecular target for an RNAi-based strategy to manage Bt resistance in Plutella xylostella (L.)

Bacillus thuringiensis (Bt), a naturally occurring entomopathogenic soil bacterium, has been the active ingredient of sprayable Bt biopesticides for over a century. Insecticidal Bt Cry proteins are particularly well suited for use as plant-incorporated protectants in transgenic crops (Bt crops) due to their specificity against insect pests and safety for non-target organisms. The sustainability of these Bt products, however, has been challenged by the development of resistance in the field. RNA interference (RNAi), a species-specific control alternative that has been deregulated and commercialized in the USA and Canada, provides a new mode of action to complement the existing Bt products. Based on our preliminary research, we hypothesized that pyramiding/integrating Bt with RNAi can address/manage resistance issues related to Bt traits. To examine this overarching hypothesis, we (1) cloned and characterized a serine/threonine kinase gene (fused) of the Hedgehog (Hh) signaling pathway in the diamondback moth, Plutella xylostella (L.), a global superpest; (2) profiled Pxfused expression in Bt-resistant P. xylostella strains; and (3) investigated the involvement of Pxfused in Bt Cry1Ac resistance in P. xylostella. Pxfused expression was elevated ubiquitously in all Bt-resistant strains, and silencing of Pxfused led to larval and pupal mortality in both Cry1Ac-susceptible and -resistant strains, suggesting that Pxfused is a potential target for RNAi-based resistance management. Taken together, our results not only identify a molecular target to control a devastating lepidopteran pest, but also shed light on a novel resistance management strategy through the integration of two biotechnological techniques with distinct modes of action.

Specific Protein 1 and p53 Interplay Modulates the Expression of the KCTD-Containing Cullin3 Adaptor Suppressor of Hedgehog 2

The Hedgehog (Hh) signaling pathway plays a crucial role in normal embryonic development and adult tissue homeostasis. On the other end, dysregulated Hh signaling triggers a prolonged mitogenic response that may prompt abnormal cell proliferation, favoring tumorigenesis. Indeed, about 30% of medulloblastomas (MBs), the most common malignant childhood cerebellar tumors, exhibit improper activation of the Hh signaling. The oncosuppressor KCASH2 has been described as a suppressor of the Hh signaling pathway, and low KCASH2 expression was observed in Hh-dependent MB tumor. Therefore, the study of the modulation of KCASH2 expression may provide fundamental information for the development of new therapeutic approaches, aimed to restore physiological KCASH2 levels and Hh inhibition. To this end, we have analyzed the TATA-less KCASH2 proximal promoter and identified key transcriptional regulators of this gene: Sp1, a TF frequently overexpressed in tumors, and the tumor suppressor p53. Here, we show that in WT cells, Sp1 binds KCASH2 promoter on several putative binding sites, leading to increase in KCASH2 expression. On the other hand, p53 is involved in negative regulation of KCASH2. In this context, the balance between p53 and Sp1 expression, and the interplay between these two proteins determine whether Sp1 acts as an activator or a repressor of KCASH2 transcription. Indeed, in p53–/– MEF and p53 mutated tumor cells, we hypothesize that Sp1 drives promoter methylation through increased expression of the DNA methyltransferase 1 (DNMT1) and reduces KCASH2 transcription, which can be reversed by Sp1 inhibition or use of demethylating agents. We suggest therefore that downregulation of KCASH2 expression in tumors could be mediated by gain of Sp1 activity and epigenetic silencing events in cells where p53 functionality is lost. This work may open new venues for novel therapeutic multidrug approaches in the treatment of Hh-dependent tumors carrying p53 deficiency.

Novel Long Noncoding RNA miR205HG Functions as an Esophageal Tumor-Suppressive Hedgehog Inhibitor.

Simple SummaryBarrett’s esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Long noncoding RNAs (lncRNAs) have been identified as key regulators of biological pathways and we identified lncRNA, miR205HG, as a tumor suppressor in the development of Barrett’s esophagus and esophageal adenocarcinoma, in part through its effect on the Hedgehog signaling pathway. The aims of the current study were: (1) to study involvement of miR205HG in the development of BE and EAC (2) to clarify the role of miR205HG in in vitro and in vivo experiments; and (3) to investigate the mechanism of miR205HG involving the Hedgehog (Hh) signaling pathwayBarrett’s esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Recently, long noncoding RNAs (lncRNAs) have been identified as key regulators of biological pathways. However, involvement of lncRNAs in the development of BE and EAC has not been well-studied. The aims of the current study were: (1) to study involvement of the lncRNA, miR205HG, in the development of BE and EAC; (2) to clarify the role of miR205HG in in vitro and in vivo experiments; and (3) to investigate the mechanism of miR205HG involving the Hedgehog (Hh) signaling pathway. These experiments revealed that miR205HG was downregulated in EAC vs. normal esophageal epithelia (NE) as well as in EAC cell lines, and its forced overexpression inhibited EAC cell proliferation and cell cycle progression in vitro. Similarly, overexpression of miR205HG inhibited xenograft tumor growth in mice In vivo. Finally, we show that one mechanism of action of miR205HG involves the Hh signaling pathway: miR205HG and Hh expression levels were inversely correlated in both EAC (r = −0.73) and BE (r = −0.83) tissues, and in vitro studies revealed details of Hh signaling inhibition induced by miR205HG. In conclusion, these findings establish that lncRNA miR205HG functions as a tumor suppressor in the development of BE and EAC, at least in part through its effect on the Hh signaling pathway.

Light-inducible control of cellular proliferation and differentiation by a Hedgehog signaling inhibitor

The Hedgehog (Hh) signaling pathway is a major regulator of cell differentiation and proliferation. Aberrant activation of the Hh pathway has been implicated in several types of cancer. To understand the Hedgehog pathway and fight against related diseases, it is important to inhibit Hedgehog signaling in a targeted manner. However, no tools are available for the precise inhibition of Hh signaling in a spatiotemporal manner. In this study, we synthesized and evaluated the bioactivity of a light-inducible Hh pathway inhibitor (NVOC-SANT-75). NVOC-SANT-75 inhibits transcription factor Gli1 in NIH3T3 cells and controls proliferation and differentiation of primary cultured mouse cerebellar neurons in a light-irradiation-dependent manner. The light-inducible Hedgehog signaling inhibitors may be a new candidate for light-mediated cancer treatment.

Recent Advances in Chemotherapeutic Implications of Deguelin: A Plant- Derived Retinoid

Deguelin, a plant retinoid has emerged to be a promising therapeutic agent in the treatment of different cancers. Recent studies demonstrate that deguelin has potential as an angiogenesis antagonist in malignant and endothelial cells by specifically targeting HGF-c-Met and VEGFVEGFR pathways. It is reported to have profound therapeutic effects in pancreatic cancer by inactivation of the hedgehog (Hh) signalling pathway and suppresses the expression of matrix metalloproteinases such as MMP-2 and MMP-9. The basic underlying mechanisms for deguelin mediated anti- NSCLC effects were uncovered through its induction of elevated intracellular Reactive Oxygen Species (ROS) levels and suppression of the PI3K /Akt-HK2 signalling pathway. Deguelin induces cell apoptosis by targeting various pathways most notably regulating the expression of galectin-1 and binding directly to anti-apoptotic Bcl-2 (B-cell lymphoma 2), Bcl-xl (B-cell lymphoma-extralarge) and Mcl-1 (Myeloid Cell Leukemia Sequence 1) in the hydrophobic grooves thereby liberating BAD and BAX from binding with these proteins. These results derived from the effect of Deguelin on various cancer cell lines have further elucidated its role as a novel anti-tumorigenic agent targeting angiogenesis, apoptosis, cell proliferation and migration for cancer chemoprevention. In this review, an attempt has been made to highlight the potential therapeutic effects of Deguelin in destroying the cancer cells by inhibiting various tumour promoting pathways and its uses as a therapeutic agent alone or in combination.

Extensive crosstalk of G protein-coupled receptors with the Hedgehog signalling pathway.

Hedgehog (Hh) ligands orchestrate tissue patterning and growth by acting as morphogens, dictating different cellular responses depending on ligand concentration. Cellular sensitivity to Hh ligands is influenced by heterotrimeric G protein activity, which controls production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). cAMP in turn activates Protein kinase A (PKA), which functions as an inhibitor and (uniquely in Drosophila) as an activator of Hh signalling. A few mammalian Gαi- and Gαs-coupled G protein-coupled receptors (GPCRs) have been shown to influence Sonic hedgehog (Shh) responses in this way. To determine whether this is a more-general phenomenon, we carried out an RNAi screen targeting GPCRs in Drosophila. RNAi-mediated depletion of more than 40% of GPCRs tested either decreased or increased Hh responsiveness in the developing Drosophila wing, closely matching the effects of Gαs and Gαi depletion, respectively. Genetic analysis indicated that the orphan GPCR Mthl5 lowers cAMP levels to attenuate Hh responsiveness. Our results identify Mthl5 as a new Hh signalling pathway modulator in Drosophila and suggest that many GPCRs may crosstalk with the Hh pathway in mammals.

Understanding the Hedgehog Signaling Pathway in Acute Myeloid Leukemia Stem Cells: A Necessary Step toward a Cure

Simple SummaryThe Hedgehog signaling pathway is related to the cell cycle. In particular, it is considered to play a fundamental role in the quiescence of leukemic stem cell (i.e., a temporary resting state without cell replication). Leukemic stem cells are the cells supposed to give rise to the relapses of the leukemia. Therefore, the Hedgehog pathway must be understood to improve the current treatments against acute myeloid leukemia and avoid the relapse of the disease. In this review, we gather the present knowledge about the physiological Hedgehog pathway function, the aberrant activation of Hedgehog in leukemia, and highlight the lack of evidence regarding some aspects of this important pathway. Finally, we summarize the acute myeloid leukemia treatments targeting this signaling pathway.A better understanding of how signaling pathways govern cell fate is fundamental to advances in cancer development and treatment. The initialization of different tumors and their maintenance are caused by the deregulation of different signaling pathways and cancer stem cell maintenance. Quiescent stem cells are resistant to conventional chemotherapeutic treatments and, consequently, are responsible for disease relapse. In this review we focus on the conserved Hedgehog (Hh) signaling pathway which is involved in regulating the cell cycle of hematopoietic and leukemic stem cells. Thus, we examine the role of the Hh signaling pathway in normal and leukemic stem cells and dissect its role in acute myeloid leukemia. We explain not only the connection between illness and the signaling pathway but also evaluate innovative therapeutic approaches that could affect the outcome of patients with acute myeloid leukemia. We found that many aspects of the Hedgehog signaling pathway remain unknown. The role of Hh has only been proven in embryo and hematopoietic stem cell development. Further research is needed to elucidate the role of GLI transcription factors for therapeutic targeting. Glasdegib, an SMO inhibitor, has shown clinical activity in acute myeloid leukemia; however, its mechanism of action is not clear.

Concomitant inhibition of hedgehog signalling and activation of retinoid receptors abolishes bleomycin-induced lung fibrosis.

Pulmonary fibrosis is a devastating disease with unknown treatment. All-trans retinoic acid (ATRA) attenuates bleomycin-induced lung fibrosis by different mechanistic pathways. However, the role of retinoid receptors in lung fibrosis is still unclear. Forskolin (FSK), a potent inhibitor for the revolutionary hedgehog (Hh) signalling pathway, has a promising antifibrotic effect on other organs such as the liver. This study investigates the interplay between the retinoid receptors modulation and the Hh signalling pathway in bleomycin (BLM)-induced pulmonary fibrosis. Rats were randomised and administrated a single dose of 7.5mg/kg of BLM alone and with ATRA, FSK and both of them. The effects of FSK and ATRA on lung functions, oxidative stress markers (malondialdehyde [MDA], glutathione [GSH], superoxide dismutase [SOD] and catalase [CAT]), retinoid markers (retinoic acid receptors [RAR] and rexinoid X receptors [RXR]) and Hh signalling markers (patched homolog 1 [Ptch-1], Smoothened [Smo] and glioblastoma-2 [Gli-2]) were assessed. In single therapies, ATRA and FSK ameliorated BLM-induced lung fibrosis. On the contrary, a combination of both drugs synergistically reversed the effect of BLM-induced lung fibrosis, as indicated by the enhancement of lung functions and the decrease of the α-smooth muscle actin (α-SMA) expression and collagen deposition. Additionally, FSK and ATRA ameliorated oxidative stress and inflammation, reduced transforming growth factor β1 (TGF-β1) levels and reversed the effect of BLM on the mRNA expression of Ptch-1, Smo and Gli-2. FSK inhibited the Hh pathway and also activated protein kinase A (PKA) that is, in part, involved in phosphorylation of RAR/RXR heterodimer (a key step in retinoid receptor activation). The present results suggest that a combination of FSK and ATRA has a promising therapeutic value for lung fibrosis management.

Construction of specific Smo lentivirus and expression of infected pancreatic cancer cells positive for CD24CD44 surface antibody.

This study aimed to construct a vector lentivirus carrying the Smo gene and transfect pancreatic cancer cells positive for CD24CD44 surface antibody and detect the infectivity. A lentivirus carrying a specific Smo fragment was designed and synthesized, and its functionality was tested. An overexpression group, inhibitory group, and negative control group were used for subsequent experimental research and comparison. A virus was successfully designed and produced. The best viral load was the 1X106 TU virus, where the cell growth and fluorescence effect of culture wells with polybrene dilution were the best. These are the transfection conditions and transfection param-eters for subsequent experiments. This plasmid was detected with a flag antibody by Western blot. The result was that it had a large specific 250kD band, and the membrane protein was overexpressed successfully. The expression results of Smo in five groups of cells after virus transfection detected by RT-PCR: blank group were 1.0038±0.0344, CON238 negative group: 1.0276±0.2944d, CON077 negative group: 0.8793±0.0402; LV-SMO15570-2 overexpres-sion group: 2.7479±0.8308, and LV-SMO-RNAi37304-1 inhibition group: 0.2386±0.0481. There were differences among the overexpression group and inhibition group with the other three groups. Homogeneity of variance: Bartlett F = 4.3530, P = 0.0016 < 0.05, heterogeneous. K-W test: cc2 = 10.9905* P = 0.0267, and there was a statisti-cally significant difference. The designed virus achieved the goal requirements. An sRNA fragment was designed for the key gene Smo of the Hh signaling pathway, and a vector lentivirus carrying this fragment was successfully constructed. The expression of Smo was analyzed after transfecting SW1990CD24CD44 positive cells, suggesting that the function of the RNA fragment designed for the key gene Smo in this experiment was successful.

Deubiquitinase USP18 regulates reactive astrogliosis by stabilizing SOX9.

Reactive astrogliosis is a pathological feature of spinal cord injury (SCI). The ubiquitin-proteasome system plays a crucial role in maintaining protein homeostasis and has been widely studied in neuroscience. Little, however, is known about the underlying function of deubiquitinating enzymes in reactive astrogliosis following SCI. Here, we found that ubiquitin-specific protease 18 (USP18) was significantly upregulated in astrocytes following scratch injury, and in the injured spinal cord in mice. Knockdown of USP18 in vitro and conditional knockout of USP18 in astrocytes (USP18 CKO) in vivo significantly attenuated reactive astrogliosis. In mice, this led to widespread inflammation and poor functional recovery following SCI. In contrast, overexpression of USP18 in mice injected with adeno-associated virus (AAV)-USP18 had beneficial effects following SCI. We showed that USP18 binds, deubiquitinates, and thus, stabilizes SRY-box transcription factor 9 (SOX9), thereby regulating reactive astrogliosis. We also showed that the Hedgehog (Hh) signaling pathway induces expression of USP18 through Gli2-mediated transcriptional activation after SCI. Administration of the Hh pathway activator SAG significantly increased reactive astrogliosis, reduced lesion area and promoted functional recovery in mice following SCI. Our results demonstrate that USP18 positively regulates reactive astrogliosis by stabilizing SOX9 and identify USP18 as a promising target for the treatment of SCI.

Molecular signatures of selection on the human GLI3 associated central nervous system specific enhancers.

The zinc finger-containing transcription factor Gli3 is a key mediator of Hedgehog (Hh) signaling pathway. In vertebrates, Gli3 has widespread expression pattern during early embryonic development. Along the anteroposterior axes of the central nervous system (CNS), dorsoventral neural pattern elaboration is achieved through Hh mediated spatio-temporal deployment of Gli3 transcripts. Previously, we and others uncovered a set of enhancers that mediate many of the known aspects of Gli3 expression during neurogenesis. However, the potential role of Gli3 associated enhancers in trait evolution has not yet received any significant attention. Here, we investigate the evolutionary patterns of Gli3 associated CNS-specific enhancers that have been reported so far. A subset of these enhancers has undergone an accelerated rate of molecular evolution in the human lineage in comparison to other primates/mammals. These fast-evolving enhancers have acquired human-specific changes in transcription factor binding sites (TFBSs). These human-unique changes within subset of Gli3 associated CNS-specific enhancers were further validated as single nucleotide polymorphisms through 1000 Genome Project Phase 3 data. This work not only infers the molecular evolutionary patterns of Gli3 associated enhancers but also provides clues for putative genetic basis of the population-specificity of gene expression regulation.

GLI1 reduces drug sensitivity by regulating cell cycle through PI3K/AKT/GSK3/CDK pathway in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy with high incidence and recurrence rates. Gene expression profiling has revealed that transcriptional overexpression of glioma‐associated oncogene 1 (GLI1), a vital gene in the Hedgehog (Hh) signaling pathway, occurs in poor-prognosis AML, and high levels of phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) and AKT3 predict shorter overall survival in AML patients. In this study, we discovered that GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity in AML cells while knocking down GLI1 has the opposite effect. Moreover, GLI1 promoted cell cycle progression and led to elevated protein levels of cyclins and cyclin-dependent kinases (CDKs) in AML cells. By luciferase assays and co-immunoprecipitation, we demonstrated that the PI3K/AKT pathway is directly activated by GLI1. GLI1 overexpression significantly accelerates tumor growth and upregulated p-AKT, CDK4, and cyclinD3 in vivo. Notably, the GLI1 inhibitor GANT61 and the CDK4/6 inhibitor PD 0332991 had synergistic effects in promoting Ara-c sensitivity in AML cell lines and patient samples. Collectively, our data demonstrate that GLI1 reduces drug sensitivity by regulating cell cycle through the PI3K/AKT/GSK3/CDK pathway, providing a new perspective for involving GLI1 and CDK4/6 inhibitors in relapsed/refractory (RR) patient treatment.

Inhibition of TGF-β1 on Gli2 expression was promoted by TNF-α in primary leukemia cells

Purpose Hedgehog (Hh) signaling pathway regulates a variety of tumors-related diseases including leukemia. Whether inhibition of TGF-β1 on Gli2 expression is promoted by TNF-α in primary leukemia cells remains to be determined. Methods Primary leukemia cells were treated with TGF-β1, TNF-α or SIS3 at different concentrations. Gli2 expression was detected by quantitative real-time PCR and western blot analyses. Results We found that TGF-β significantly decreased Gli2 expression, and co-treatment with TNF-αfurther decreased Gli2 expression in primary leukemia cells. TNF-α can increased TGF-βRI and TGF-βRII protein expression in primary leukemia cells, while SIS3 inhibited the effect of TGF-β. Conclusion Our results suggest that Gli2 expression in primary leukemia cells is induced by TGF-β in a Smad3-dependent manner, and independent of Hh receptor signaling.