Activation Of Sonic Hedgehog Signaling Research Articles

Activation of Sonic hedgehog signal by Purmorphamine, in a mouse model of Parkinson's disease, protects dopaminergic neurons and attenuates inflammatory response by mediating PI3K/AKt signaling pathway.

In Parkinson's disease (PD), microglial activation-mediated neuroinflammation is associated with dopaminergic neurons degeneration in the substantia nigra pars compacta. Previous studies that have investigated this neurodegenerative disease have reported that the Sonic hedgehog (SHH) signaling pathway, through inhibiting the inflammatory processes, exerts a beneficial neuroprotective effect. However, the mechanisms underlying the anti-inflammatory and neuroprotective effects of this signaling pathway remain poorly understood. The present study aimed to further investigate these mechanisms in vitro and in vivo. At first, BV2 microglial cells treated with lipopolysaccharide (LPS) were used to induce an inflammatory response. It was observed that the activation of SHH signaling by Purmorphamine attenuated the LPS-induced inflammatory response, increased the expression of transforming growth factor-β1 through the phosphatidylinositol 3-kinase (PI3K)/AKT serine/threonine kinase (Akt) intracellular signaling pathway and inhibited nuclear receptor subfamily 4 group A member 2, independently of the PI3K/Akt signaling pathway. Furthermore, the blockade of the PI3K/Akt signaling pathway by intranasal administration of LY294002, significantly reduced the SHH-associated neuroprotective effects on dopaminergic neurons, improved motor functions, and increased the microglial activation and inflammatory response in a mouse model of PD induced using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In conclusion, the data of the present study reported that anti-inflammatory and neuroprotective effects can be obtained in BV2 microglial cells and in a mouse model of PD by successive activation of the SHH and PI3K/Akt signaling pathways.

Could an endoneurial endothelial crosstalk between Wnt/β-catenin and Sonic Hedgehog pathways underlie the early disruption of the infra-orbital blood-nerve barrier following chronic constriction injury?

BackgroundBlood–nerve barrier disruption is pivotal in the development of neuroinflammation, peripheral sensitization, and neuropathic pain after peripheral nerve injury. Activation of toll-like receptor 4 and inactivation of Sonic Hedgehog signaling pathways within the endoneurial endothelial cells are key events, resulting in the infiltration of harmful molecules and immunocytes within the nerve parenchyma. However, we showed in a previous study that preemptive inactivation of toll-like receptor 4 signaling or sustained activation of Sonic Hedgehog signaling did not prevent the local alterations observed following peripheral nerve injury, suggesting the implication of another signaling pathway.MethodsUsing a classical neuropathic pain model, the infraorbital nerve chronic constriction injury (IoN-CCI), we investigated the role of the Wnt/β-catenin pathway in chronic constriction injury-mediated blood–nerve barrier disruption and in its interactions with the toll-like receptor 4 and Sonic Hedgehog pathways. In the IoN-CCI model versus control, mRNA expression levels and/or immunochemical detection of major Wnt/Sonic Hedgehog pathway (Frizzled-7, vascular endothelial-cadherin, Patched-1 and Gli-1) and/or tight junction proteins (Claudin-1, Claudin-5, and Occludin) readouts were assessed. Vascular permeability was assessed by sodium fluorescein extravasation.ResultsIoN-CCI induced early alterations in the vascular endothelial-cadherin/β-catenin/Frizzled-7 complex, shown to participate in local blood–nerve barrier disruption via a β-catenin-dependent tight junction protein downregulation. Wnt pathway also mediated a crosstalk between toll-like receptor 4 and Sonic Hedgehog signaling within endoneurial endothelial cells. Nevertheless, preemptive inhibition of Wnt/β-catenin signaling before IoN-CCI could not prevent the downregulation of key Sonic Hedgehog pathway readouts or the disruption of the infraorbital blood–nerve barrier, suggesting that Sonic Hedgehog pathway inhibition observed following IoN-CCI is an independent event responsible for blood–nerve barrier disruption.ConclusionA crosstalk between Wnt/β-catenin- and Sonic Hedgehog-mediated signaling pathways within endoneurial endothelial cells could mediate the chronic disruption of the blood–nerve barrier following IoN-CCI, resulting in increased irreversible endoneurial vascular permeability and neuropathic pain development.

Molecular Classification of Hepatocellular Adenoma Associates With Risk Factors, Bleeding, and Malignant Transformation

Hepatocellular adenomas (HCAs) are benign liver tumors that can be assigned to molecular subtypes based on inactivating mutations in hepatocyte nuclear factor 1A, activating mutations in β-catenin, or activation of inflammatory signaling pathways. We aimed to update the classification system for HCA and associate the subtypes with disease risk factors and complications. We analyzed expression levels of 20 genes and sequenced exon regions of 8 genes (HNF1A, IL6ST, CTNNB1, FRK, STAT3, GNAS, JAK1, and TERT) in 607 samples of 533 HCAs from 411 patients, collected from 28 centers mainly in France from 2000 and 2014. We performed gene expression profile, RNA sequence, whole-exome and genome sequence, and immunohistochemical analyses of select samples. Molecular data were associated with risk factors, histopathology, bleeding, and malignant transformation. Symptomatic bleeding occurred in 14% of the patients (85% of cases were female, median age, 38 years); 7% of the nodules were borderline between HCA and hepatocellular carcinoma, and 3% of patients developed hepatocellular carcinoma from HCA. Based on molecular features, we classified HCA into 8 subgroups. One new subgroup, composed of previously unclassified HCA, represented 4% of HCAs overall and was associated with obesity and bleeding. These tumors were characterized by activation of sonic hedgehog signaling, due to focal deletions that fuse the promoter of INHBE with GLI1. Analysis of genetic heterogeneity among multiple HCAs, from different patients, revealed a molecular subtype field effect; multiple tumors had different mutations that deregulated similar pathways. Specific molecular subtypes of HCA associated with various HCA risk factors, including imbalances in estrogen or androgen hormones. Specific molecular subgroup of HCA with β-catenin and sonic hedgehog activation associated with malignant transformation and bleeding, respectively. Using sequencing and gene expression analyses, we identified a subgroup of HCA characterized by fusion of the INHBE and GLI1 genes and activation of sonic hedgehog pathway. Molecular subtypes of HCAs associated with different patients' risk factors for HCA,disease progression, and pathology features of tumors. This classification system might be used to select treatment strategies for patients withHCA.

Hedgehog Controls Quiescence and Activation of Neural Stem Cells in the Adult Ventricular-Subventricular Zone.

SummaryIdentifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury.

Abstract 4556: Activation of sonic hedgehog signaling is essential for non-alcoholic steatohepatitis induced by liver-specific disruption of Nrf1

Abstract Liver-specific inactivation of NF-E2 p45-related factor 1 (Nrf1) results in sequential development of non-alcoholic steatohepatitis (NASH), fibrosis, and finally hepatocellular carcinoma (HCC). However, the detailed mechanisms responsible for this phenotype remain uncertain. In the present study, a β-naphthoflavone (β-NF)-inducible liver-specific Nrf1 knockout mouse line which harbors an Nrf1flox/flox allele and a rat CYP1A1-Cre transgene was employed to investigate potential mechanism underlying Nrf1 deficiency-induced NASH. β-NF-induced liver-specific knockout Nrf1 resulted in a reversible typical NASH and mild fibrosis phenotype. β-NF treatment almost abolished Nrf1 expression after 2 weeks, and induced dramatic elevation of serum transaminase, inflammation, necrosis, ballooning degeneration and HSC activation, but these phenotype diminished after 16 weeks, accompanied by restoration of Nrf1 expression. These observations clearly demonstrated the dependency of NASH and fibrosis on loss of Nrf1, and provide a novel model of reversible non-alcoholic liver diseases. Notably, disruption of Nrf1 caused a sustained activation of sonic hedgehog (shh) signaling pathway accompanied by pronounced endoplasmic reticulum (ER) stress. More importantly, vismodegib, a hedgehog signaling inhibitor, dramatically reversed the deleterious effects caused by loss of Nrf1, such as elevation of serum transaminase activities, inflammatory cells infiltration and hepatocyte necrosis. It has been reported that hepatocytes undergoing endoplasmic reticulum stress and showed elevated expression of hedgehog ligands. However, the mRNA level of hedgehog ligand was not changed in Nrf1 deficiency mice, while the protein levels of both shh and Gli2 were significantly increased. Furthermore, chemical chaperone 4-PBA which can promote protein degradation partially reversed the effects of Nrf1 deficiency on shh signaling. Collectively, these data demonstrate that activation of sonic hedgehog signaling is essential for non-alcoholic steatohepatitis induced by liver-specific disruption of Nrf1, possibly through post-translational modification/procession of shh, and hedgehog signaling could be targeted to treat non-alcoholic liver diseases. The present works were supported by the National Natural Science Foundation of China (No. 91429305, 81272468, and 81372266) Citation Format: Mingnan Cao, Limei Guo, Juan Li, Chun Liu, Yiguo Zhang, Siwang Yu, Tony A-N. Kong. Activation of sonic hedgehog signaling is essential for non-alcoholic steatohepatitis induced by liver-specific disruption of Nrf1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4556.

Sonic hedgehog-mediated epithelial-mesenchymal transition in renal tubulointerstitial fibrosis.

The sonic hedgehog (SHH) signaling pathway plays a critical role in embryonic development, tissue regeneration and organogenesis. The activation of SHH signaling produces profibrogenic effects in various tissues, such as the liver and the biliary ducts. However, the role of SHH signaling in renal fibrogenesis remains to be elucidated. For this purpose, in the present study, we evaluated the hypothesis that activated SHH signaling promotes the acquisition of a myofibroblastic phenotype through the epithelial-mesenchymal transition (EMT), resulting in renal interstitial fibrosis (RIF). Kidney samples from rats subjected to unilateral or bilateral ureteral obstruction exhibited the enhanced expression of SHH-pathway proteins, mesenchymal markers and the decreased expression of epithelial markers. Overactive SHH signaling as well as tubular EMT and RIF in the obstructed kidneys were inhibited by recanalization of the ureter. In vitro, SHH signaling was activated during EMT induction and extracellular matrix (ECM) deposition was observed in transforming growth factor-β1 (TGF-β1)-treated renal tubular epithelial cells [RTECs; NRK-52E cell line]. Exogenous SHH activated SHH signaling and resulted in the upregulated expression of mesenchymal genes, the profibrogenic cytokine TGF-β1, and the downregulated expression of epithelial markers. The blockade of SHH signaling with cyclopamine abolished SHH-mediated EMT as well as the acquisition of a myofibroblastic phenotype, and decreased TGF-β1 expression and ECM production. Thus, taken together, these findings demonstrate that the activation of the SHH signaling pathway promotes the induction of EMT and renal tubulointerstitial fibrosis. The pharmacological inhibition of SHH signaling may potentially be of therapeutic value in the management of fibrotic kidney diseases.

Abstract IA03: Insights into the origins and pathogenesis of embryonal rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive chemotherapy, radiotherapy and surgery, clinical outcomes for RMS have not improved for three decades, emphasizing the need to uncover the molecular underpinnings of the disease. RMS is an aggressive skeletal muscle-lineage tumor composed of malignant myoblasts that fail to exit the cell cycle and are blocked from fusing into syncytial muscle. RMS includes two histolopathologic subtypes: alveolar RMS, driven by the fusion protein PAX3/7-FOXO1, and embryonal RMS (ERMS), which is genetically heterogeneous. RMS has been presumed to originate from derailed muscle progenitors based on the histologic appearance and gene expression pattern of the tumors. However, an origin restricted to skeletal muscle does not explain RMS occurring in tissues devoid of skeletal muscle such as the prostate, bladder, biliary tree and the omentum. Previously, we showed that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of an adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human ERMS. In this model, tumorigenesis occurs with high penetrance (~80%), is early onset (by 2 months of age), and is restricted to the head and neck. Also, unlike previous RMS models, this model requires no additional background mutations, such as inactivation of p53, and drives only ERMS neoplasia. We illustrated that the transcriptome of the aP2-Cre;SmoM2 tumors recapitulates both other mouse ERMS models as well as human ERMS. With the short latency and anatomic restricted tumor location, we sought to leverage this model to explore the cell of origin. Lineage tracing the aP2-Cre with reporter mice illustrated aP2-Cre expression in both brown and white adipose tissue as well as a discrete population of cells lying between skeletal muscle fibers but not beneath the laminin sheath of the muscle fibers. These aP2-lineage cells are distinct from Pax7-positive skeletal muscle stem cells or satellite cells. The aP2-lineage cells do not contribute to myotube formation in vitro. When compared to aP2-Cre;R26-Tom mice, the addition of oncogenic SmoM2 (aP2-Cre;R26-Tom;SmoM2) results in embryonic expansion of the aP2-lineage interstitial muscle cells. Our findings suggest that non-skeletal muscle progenitors are a potential cell of origin for Sonic Hedgehog-driven ERMS. Citation Format: Catherine J. Drummond, Daniel Devine, Matthew R. Garcia, Mark E. Hatley. Insights into the origins and pathogenesis of embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA03.

GALNT1-Mediated Glycosylation and Activation of Sonic Hedgehog Signaling Maintains the Self-Renewal and Tumor-Initiating Capacity of Bladder Cancer Stem Cells.

The existence of bladder cancer stem cells (BCSC) has been suggested to underlie bladder tumor initiation and recurrence. Sonic Hedgehog (SHH) signaling has been implicated in promoting cancer stem cell (CSC) self-renewal and is activated in bladder cancer, but its impact on BCSC maintenance is unclear. In this study, we generated a mAb (BCMab1) against CD44(+) human bladder cancer cells that recognizes aberrantly glycosylated integrin α3β1. The combination of BCMab1 with an anti-CD44 antibody identified a BCMab1(+)CD44(+) cell subpopulation as BCSCs with stem cell-like properties. Gene expression analysis revealed that the hedgehog pathway was activated in the BCMab1(+)CD44(+) subpopulation and was required for BCSC self-renewal. Furthermore, the glycotransferase GALNT1 was highly expressed in BCMab1(+)CD44(+) cells and correlated with clinicopathologic features of bladder cancers. Mechanistically, GALNT1 mediated O-linked glycosylation of SHH to promote its activation, which was essential for the self-renewal maintenance of BCSCs and bladder tumorigenesis. Finally, intravesical instillation of GALNT1 siRNA and the SHH inhibitor cyclopamine exerted potent antitumor activity against bladder tumor growth. Taken together, our findings identify a BCSC subpopulation in human bladder tumors that appears to be responsive to the inhibition of GALNT1 and SHH signaling, and thus highlight a potential strategy for preventing the rapid recurrence typical in patients with bladder cancer.

Sonic hedgehog inhibitors prevent colitis-associated cancer via orchestrated mechanisms of IL-6/gp130 inhibition, 15-PGDH induction, Bcl-2 abrogation, and tumorsphere inhibition

Sonic hedgehog (SHH) signaling is essential in normal development of the gastrointestinal (GI) tract, whereas aberrantly activated SHH is implicated in GI cancers because it facilitates carcinogenesis by redirecting stem cells. Since colitis-associated cancer (CAC) is associated with inflammatory bowel diseases, in which SHH and IL-6 signaling, inflammation propagation, and cancer stem cell (CSC) activation have been implicated, we hypothesized that SHH inhibitors may prevent CAC by blocking the above SHH-related carcinogenic pathways. In the intestinal epithelial cells IEC-6 and colon cancer cells HCT-116, IL-6 expression and its signaling were assessed with SHH inhibitors and levels of other inflammatory mediators, proliferation, apoptosis, tumorsphere formation, and tumorigenesis were also measured. CAC was induced in C57BL/6 mice by administration of azoxymethane followed by dextran sodium sulfate administration. SHH inhibitors were administered by oral gavage and the mice were sacrificed at 16 weeks. TNF-α–stimulated IEC-6 cells exhibited increased levels of proinflammatory cytokines and enzymes, whereas SHH inhibitors suppressed TNF-α–induced inflammatory signaling, especially IL-6/IL-6R/gp130 signaling. SHH inhibitors significantly induced apoptosis, inhibited cell proliferation, suppressed tumorsphere formation, and reduced stemness factors. In the mouse model, SHH inhibitors significantly reduced tumor incidence and multiplicity, decreased the expression of IL-6, TNF-α, COX-2, STAT3, and NF-κB, and significantly induced apoptosis. In colosphere xenografts, SHH inhibitor significantly suppressed tumorigenesis by inhibiting tumorsphere formation. Taken together, our data suggest that administration of SHH inhibitors could be an effective strategy to prevent colitis-induced colorectal carcinogenesis, mainly by targeting IL-6 signaling, ablating CSCs, and suppressing oncogenic inflammation, achieving chemoquiescence ultimately.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Glial Sonic hedgehog signaling activity is required for proper cortical expansion and cerebellar architecture

Glial Sonic hedgehog signaling activity is required for proper cortical expansion and cerebellar architecture

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells.

Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.

Corrigendum] Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma.

Mol Med Rep 12: [Related article:] 6702–6710, 2015; DOI: 10.3892/mmr.2015.4229 After the publication of the article, it has been brought to the authors' attention by an interested reader that we had made an error regarding the presentation of certain data in the manuscript. The error relates to the presentation of Figs. 1 and 2 in the paper: The control panels for Fig. 1C [labelled 'cyclopamine (µM)'] and Figs. 2B and C [labelled 'rhSSH (µg/ml)'] were derived from the same image. The control U251 cells, featured in Fig. 1 and Figs. 2B and C, were treated without cyclopamine and rhSHH. Therefore, the U251 cells treated without cyclopamine and rhSHH were considered as a control group compared with U251 cells that were separately treated with cyclopamine or rhSHH, and these were photographed randomly. A new Fig. 2 is provided, which contains the correct data for the control panels for Figs. 2B and C.

Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma.

Aberrant hedgehog signaling contributes to the development of various malignancies, including glioblastoma (GBM). However, the potential mechanism of hedgehog signaling in GBM migration and invasion has remained to be elucidated. The present study showed that enhanced hedgehog signaling by recombinant human sonic hedgehog N-terminal peptide (rhSHH) promoted the adhesion, invasion and migration of GBM cells, accompanied by increases in mRNA and protein levels of matrix metalloproteinase-2 (MMP-2) and MMP-9. However, inhibition of hedgehog signaling with cyclopamine suppressed the adhesion, invasion and migration of GBM cells, accompanied by decreases in mRNA and protein levels of MMP-2 and -9. Furthermore, it was found that MMP-2- and MMP-9-neutralizing antibodies or GAM6001 reversed the inductive effects of rhSHH on cell migration and invasion. In addition, enhanced hedgehog signaling by rhSHH increased AKT phosphorylation, whereas blockade of hedgehog signaling decreased AKT phosphorylations. Further experiments showed that LY294002, an inhibitor of phosphoinositide-3 kinase (PI3K), decreased rhSHH-induced upregulation of MMP-2 and -9. Finally, the protein expression of glioblastoma-associated oncogene 1 was positively correlated with levels of phosphorylated AKT as well as protein expressions of MMP-2 and -9 in GBM tissue samples. In conclusion, the present study indicated that the hedgehog pathway regulates GBM-cell migration and invasion by increasing MMP-2 and MMP-9 production via the PI3K/AKT pathway.

Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts cardioprotection against ischemia reperfusion injuries.

Sonic hedgehog (SHH) is a conserved protein involved in embryonic tissue patterning and development. SHH signaling has been reported as a cardio-protective pathway via muscle repair–associated angiogenesis. The goal of this study was to investigate the role of SHH signaling pathway in the adult myocardium in physiological situation and after ischemia-reperfusion. We show in a rat model of ischemia-reperfusion that stimulation of SHH pathway, either by a recombinant peptide or shed membranes microparticles harboring SHH ligand, prior to reperfusion reduces both infarct size and subsequent arrhythmias by preventing ventricular repolarization abnormalities. We further demonstrate in healthy animals a reduction of QTc interval mediated by NO/cGMP pathway leading to the shortening of ventricular cardiomyocytes action potential duration due to the activation of an inward rectifying potassium current sharing pharmacological and electrophysiological properties with ATP-dependent potassium current. Besides its effect on both angiogenesis and endothelial dysfunction we demonstrate here a novel cardio-protective effect of SHH acting directly on the cardiomyocytes. This emphasizes the pleotropic effect of SHH pathway as a potential cardiac therapeutic target.

Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

Glioblastoma Multiforme (GBM), the most common and lethal adult primary tumor of the brain, showed a link between Sonic Hedgehog (SHH) pathway in the resistance to temozolomide (TMZ). PTCH1, the SHH receptor, can tonically represses signaling by endocytosis. We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance. TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein. Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and ectopic expression identified miR-9 as the target of PTCH1 in resistant GBM cells with concomitant activation of SHH signaling. MiR-9 mediated increases in the drug efflux transporters, MDR1 and ABCG2. MiR-9 was increased in the tissues from GBM patients and in an early passage GBM cell line from a patient with recurrent GBM but not from a naïve patient. Pharmacological inhibition of SHH signaling sensitized the GBM cells to TMZ. Taken together, miR-9 targets PTCH1 in GBM cells by a SHH-independent method in GBM cells for TMZ resistance. The identified pathways could lead to new strategies to target GBM with combinations of drugs.

Differentiation of hypothalamic-like neurons from human pluripotent stem cells.

The hypothalamus is the central regulator of systemic energy homeostasis, and its dysfunction can result in extreme body weight alterations. Insights into the complex cellular physiology of this region are critical to the understanding of obesity pathogenesis; however, human hypothalamic cells are largely inaccessible for direct study. Here, we developed a protocol for efficient generation of hypothalamic neurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) obtained from patients with monogenetic forms of obesity. Combined early activation of sonic hedgehog signaling followed by timed NOTCH inhibition in human ESCs/iPSCs resulted in efficient conversion into hypothalamic NKX2.1+ precursors. Application of a NOTCH inhibitor and brain-derived neurotrophic factor (BDNF) further directed the cells into arcuate nucleus hypothalamic-like neurons that express hypothalamic neuron markers proopiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AGRP), somatostatin, and dopamine. These hypothalamic-like neurons accounted for over 90% of differentiated cells and exhibited transcriptional profiles defined by a hypothalamic-specific gene expression signature that lacked pituitary markers. Importantly, these cells displayed hypothalamic neuron characteristics, including production and secretion of neuropeptides and increased p-AKT and p-STAT3 in response to insulin and leptin. Our results suggest that these hypothalamic-like neurons have potential for further investigation of the neurophysiology of body weight regulation and evaluation of therapeutic targets for obesity.

CS-38 * BAI1 IS A NEW TUMOR SUPPRESSOR IN MEDULLOBLASTOMA

Brain-specific Angiogenesis Inhibitor 1 (BAI1) is a seven transmembrane G protein-coupled receptor (GPCR) with potent anti-angiogenic and anti-tumorigenic properties in gliomas. Our novel findings show that BAI1 expression is significantly reduced in human medulloblastoma (MB) by epigenetic mechanisms, leading us to hypothesize that loss of expression of BAI1 may favor tumor development during cerebellar development. To test this hypothesis, we generated a Bai1 knockout (KO) mouse and detected aberrant activation of Sonic hedgehog signaling and a thicker external granular layer during early postnatal cerebellum development. Furthermore, upon crossing the Bai1 KO mice with mice heterozygous for patched 1 (ptc1 + /-), we found that deficiency of Bai1 dramatically accelerated MB tumorigenesis, the first demonstration that a reduction in Bai1 dosage can promote MB formation in vivo. Moreover, restoration of BAI1 expression in human MB cells inhibited cell growth both in vitro and in vivo. These results support that BAI1 is a novel tumor suppressor for MB and that down-regulation of BAI1 in the cerebellum may drive inappropriate downstream growth signaling and contribute to neoplastic transformation leading to the formation of MB. To further elucidate the molecular mechanisms, we are currently exploring the Bai1 downstream signaling involved in MB tumorigenesis and will present the related findings. Our results also suggest that restoration of BAI1 expression may represent a novel therapeutic intervention for MB. Specifically, we demonstrate that small molecule epigenetic inhibitors of either EZH2 or MBD2 can reactivate BAI1 expression. Taken together, our novel findings provide insight into the neurobiological mechanisms underlying MB tumorigenesis and provide the rationale for the use of epigenetic therapeutics against MB.

Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-α-induced apoptosis.

BackgroundIncreased incidence of lung cancer among pulmonary tuberculosis patients suggests mycobacteria-induced tumorigenic response in the host. The alveolar epithelial cells, candidate cells that form lung adenocarcinoma, constitute a niche for mycobacterial replication and infection. We thus explored the possible mechanism of M. bovis Bacillus Calmette-Guérin (BCG)-assisted tumorigenicity in type II epithelial cells, human lung adenocarcinoma A549 and other cancer cells.MethodsCancer cell lines originating from lung, colon, bladder, liver, breast, skin and cervix were treated with tumor necrosis factor (TNF)-α in presence or absence of BCG infection. p53, COP1 and sonic hedgehog (SHH) signaling markers were determined by immunoblotting and luciferase assays, and quantitative real time PCR was done for p53-responsive pro-apoptotic genes and SHH signaling markers. MTT assays and Annexin V staining were utilized to study apoptosis. Gain- and loss-of-function approaches were used to investigate the role for SHH and COP1 signaling during apoptosis. A549 xenografted mice were used to validate the contribution of BCG during TNF-α treatment.ResultsHere, we show that BCG inhibits TNF-α-mediated apoptosis in A549 cells via downregulation of p53 expression. Substantiating this observation, BCG rescued A549 xenografts from TNF-α-mediated tumor clearance in nude mice. Furthermore, activation of SHH signaling by BCG induced the expression of an E3 ubiquitin ligase, COP1. SHH-driven COP1 targeted p53, thereby facilitating downregulation of p53-responsive pro-apoptotic genes and inhibition of apoptosis. Similar effects of BCG could be shown for HCT116, T24, MNT-1, HepG2 and HELA cells but not for HCT116 p53-/- and MDA-MB-231 cells.ConclusionOur results not only highlight possible explanations for the coexistence of pulmonary tuberculosis and lung cancer but also address probable reasons for failure of BCG immunotherapy of cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-210) contains supplementary material, which is available to authorized users.

Genetic Activation of Hedgehog Signaling Unbalances the Rate of Neural Stem Cell Renewal by Increasing Symmetric Divisions

SummaryIn the adult brain, self-renewal is essential for the persistence of neural stem cells (NSCs) throughout life, but its regulation is still poorly understood. One NSC can give birth to two NSCs or one NSC and one transient progenitor. A correct balance is necessary for the maintenance of germinal areas, and understanding the molecular mechanisms underlying NSC division mode is clearly important. Here, we report a function of the Sonic Hedgehog (SHH) receptor Patched in the direct control of long-term NSC self-renewal in the subependymal zone. We show that genetic conditional activation of SHH signaling in adult NSCs leads to their expansion and the depletion of their direct progeny. These phenotypes are associated in vitro with an increase in NSC symmetric division in a process involving NOTCH signaling. Together, our results demonstrate a tight control of adult neurogenesis and NSC renewal driven by Patched.