Shh Activity Research Articles

Primary cilia shape postnatal astrocyte development through Sonic Hedgehog signaling.

Primary cilia function as specialized signaling centers that regulate many cellular processes including neuron and glia development. Astrocytes possess cilia, but the function of cilia in astrocyte development remains largely unexplored. Critically, dysfunction of either astrocytes or cilia contributes to molecular changes observed in neurodevelopmental disorders. Here, we show that a sub-population of developing astrocytes in the prefrontal cortex are ciliated. This population corresponds to proliferating astrocytes and largely expresses the ciliary protein ARL13B. Genetic ablation of astrocyte cilia in vivo at two distinct stages of astrocyte development results in changes to Sonic Hedgehog (Shh) transcriptional targets. We show that Shh activity is decreased in immature and mature astrocytes upon loss of cilia. Furthermore, loss of cilia in immature astrocytes results in decreased astrocyte proliferation and loss of cilia in mature astrocytes causes enlarged astrocyte morphology. Together, these results indicate that astrocytes require cilia for Shh signaling throughout development and uncover functions for astrocyte cilia in regulating astrocyte proliferation and maturation. This expands our fundamental knowledge of astrocyte development and cilia function to advance our understanding of neurodevelopmental disorders.

G-quadruplexes are a source of vulnerability in BRCA2 deficient granule cell progenitors and medulloblastoma.

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 causes Fanconi anemia, complementation group FA-D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under SHH activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2 Δex3-4 ;Trp53 -/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping G-quadruplexes (G4s). Brca2-deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary MB tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2-deficient medulloblastomas.

Medulloblastoma in children with Fanconi anemia: Association with FA-D1/FA-N, SHH type and poor survival independent of treatment strategies.

Outcome of children with medulloblastoma (MB) and Fanconi Anemia (FA), an inherited DNA repair deficiency, has not been described systematically. Treatment is complicated by high vulnerability to treatment-associated side effects, yet structured data are lacking. This study aims at giving a comprehensive overview about clinical and molecular characteristics of pediatric FA MB patients. Clinical data including detailed information on treatment and toxicities of six previously unreported FA MB patients were supplemented with data of 16 published cases. We identified 22 cases of children with FA and MB with clinical data available. All MBs with subgroup reporting were SHH-activated (n=9), confirmed by methylation profiling in five patients. FA MB patients exclusively belonged to complementation groups FA-D1 (n=16) or FA-N (n=3). Patients were treated with postoperative chemotherapy only (50%) or radiotherapy (RT)±chemotherapy (27%). 23% did not receive adjuvant therapy. Excessive treatment-related toxicities were frequent. Severe hematological toxicity occurred in 91% of patients treated with alkylating chemotherapy, while non-alkylating agents and RT were less toxic. Median overall survival (OS) was 1 year (95%CI 0.3-1.8). 1-year-progression-free-survival (PFS) was 26.3±10.1% and 1-year-OS was 42.1±11.3%. Adjuvant therapy prolonged survival (1y-OS/1y-PFS 0%/0% without adjuvant therapy vs. 53.3±12.9%/33.3±12.2% with adjuvant therapy, p=0.006/p=0.086). MB in FA patients is strongly associated with SHH activation and FA-D1/FA-N. Despite the dismal prognosis, adjuvant therapy may prolong survival. Non-alkylating chemotherapy and RT are feasible in selected patients with careful monitoring of toxicities and dose adjustments. Curative therapy for FA MB-SHH remains an unmet medical need.

NFS-01. EXTREMELY RARE CASE OF CHILDREN WITH NIJMEGEN BREAKAGE SYNDROME AND MEDULLOBLASTOMA – A THERAPEUTIC CHALLENGE

Abstract BACKGROUND Nijmegen Breakage Syndrome (NBS) is an autosomal recessive inherited disorder characterized by DNA repair abnormalities. It manifests with heightened sensitivity to radiotherapy (RT) and predisposes individuals to neoplastic diseases. Antineoplastic treatment in NBS patients is particularly challenging due to concurrent immune disorders, intense toxicities, and contraindications for RT. Hematologic disorders, especially lymphomas, are among the most prevalent malignancies in NBS patients. Solid tumors in NBS are extremely rare, with only two pediatric cases documented in the literature, both succumbing due to severe complications of treatment. METHODS A review and analysis of the medical documentation of a child with NBS and MB treated at the Department of Pediatrics, Hematology, and Oncology, Gdansk, Poland, were conducted. RESULTS A genetically confirmed NBS girl displaying typical phenotypic features presents symptoms at the age of 2 years, such as headaches, reluctance to walk, and drowsiness. Imaging confirmed a tumor in the left cerebellar hemisphere, measuring 40x33x30mm, displaying radiological features consistent with Medulloblastoma (MB). The patient underwent gross total resection of the tumor. The pathology revealed a desmoplastic type of MB with SHH activation, TP53 wild type, and ongoing molecular analysis. Postoperative imaging confirmed clinical remission, and cerebrospinal fluid examination was negative. Considering the substantial toxicities of chemotherapy in NBS patients and the contraindications for RT, the patient received chemotherapy with reduced cytostatic doses. Treatment was complicated by profound and prolonged myelosuppression and infections. Currently, after completing six cycles of chemotherapy, the patient is still in remission. To maintain remission and address NBS, the patient will receive allogeneic bone marrow transplantation from a 9/10 HLA-matched unrelated donor. CONCLUSIONS Managing patients with NBS, complicated by neoplastic diseases, is exceptionally demanding and necessitates an individualized multidisciplinary approach. Modern diagnostic and therapeutic methods enhance the prospects of reducing the toxicity of oncologic treatment, improving quality of life, and extending overall survival.

NFS-03. MEDULLOBLASTOMA IN CHILDREN WITH FANCONI ANEMIA: ASSOCIATION WITH FA-D1/FA-N, SHH TYPE AND POOR SURVIVAL INDEPENDENT OF TREATMENT STRATEGIES

Abstract BACKGROUND Outcome of children with medulloblastoma (MB) and Fanconi Anemia (FA), an inherited DNA repair deficiency, has not systematically been described. Treatment is complicated by high vulnerability to treatment-associated side effects, yet structured data are lacking. This study provides a comprehensive overview about clinical and molecular characteristics of pediatric FA MB patients. METHODS Clinical data including detailed information on treatment and toxicities of six previously unreported FA MB patients were supplemented with data of 16 published cases. RESULTS We identified 22 cases of children with FA and MB with clinical data available. Biological subgroup was SHH in all cases with data available (n=9), confirmed by methylation profiling in five patients. FA MB patients exclusively belonged to complementation groups FA-D1 (n=16) or FA-N (n=3). Patients were treated with postoperative chemotherapy only (50%) or radiotherapy (RT)±chemotherapy (27%). 23% did not receive adjuvant therapy. Excessive treatment-related toxicities were frequent. Severe hematological toxicity occurred in 91% of patients treated with alkylating chemotherapy, while non-alkylating agents and RT were less toxic. 14 patients (63.6%) developed 20 other malignancies, of which ten occurred before, five simultaneously with and five after MB diagnosis. Median overall survival (OS) was 1 year (95%CI 0.3-1.9). 1-year-progression-free-survival (PFS) was 26.3±10.1% and 1-year-OS was 42.1±11.3%. Adjuvant therapy prolonged survival (1y-OS/1y-PFS 0%/0% without adjuvant therapy vs. 53.3±12.9%/33.3±12.2% with adjuvant therapy, p=0.006/p=0.086), with no difference whether the patient had received chemotherapy only or RT±chemotherapy. CONCLUSIONS MB in FA patients is strongly associated with SHH activation and FA-D1/FA-N. Despite the dismal prognosis, adjuvant therapy may improve survival. Non-alkylating chemotherapy and RT are feasible in selected patients with careful monitoring of toxicities and dose adjustments. Unlike in standard MB protocols, focal RT may be considered in FA MB patients. Curative therapy for FA MB-SHH remains an unmet medical need.

Astrocyte modulation of synaptic plasticity mediated by activity-dependent Sonic hedgehog signaling.

The influence of neural activity on astrocytes and their reciprocal interactions with neurons has emerged as an important modulator of synapse function. Astrocytes exhibit activity-dependent changes in gene expression, yet the molecular mechanisms by which they accomplish this have remained largely unknown. The molecular signaling pathway, Sonic hedgehog (Shh), mediates neuron-astrocyte communication and regulates the organization of cortical synapses. Here, we demonstrate that neural activity stimulates Shh signaling in cortical astrocytes and upregulates expression of Hevin and SPARC, astrocyte derived molecules that modify synapses. Whisker stimulation and chemogenetic activation both increase Shh activity in deep layers of the somatosensory cortex, where neuron-astrocyte Shh signaling is predominantly found. Experience-dependent Hevin and SPARC require intact Shh signaling and selective loss of pathway activity in astrocytes occludes experience-dependent structural plasticity. Taken together, these data identify Shh signaling as an activity-dependent, neuronal derived cue that stimulates astrocyte interactions with synapses and promotes synaptic plasticity.

TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.

A Spatio-Temporal-Dependent Requirement of Sonic Hedgehog in the Early Development of Sclerotome-Derived Vertebrae and Ribs.

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations.

Three-dimensional liquid metal-based neuro-interfaces for human hippocampal organoids

Human hippocampal organoids (hHOs) derived from human induced pluripotent stem cells (hiPSCs) have emerged as promising models for investigating neurodegenerative disorders, such as schizophrenia and Alzheimer’s disease. However, obtaining the electrical information of these free-floating organoids in a noninvasive manner remains a challenge using commercial multi-electrode arrays (MEAs). The three-dimensional (3D) MEAs developed recently acquired only a few neural signals due to limited channel numbers. Here, we report a hippocampal cyborg organoid (cyb-organoid) platform coupling a liquid metal-polymer conductor (MPC)-based mesh neuro-interface with hHOs. The mesh MPC (mMPC) integrates 128-channel multielectrode arrays distributed on a small surface area (~2*2 mm). Stretchability (up to 500%) and flexibility of the mMPC enable its attachment to hHOs. Furthermore, we show that under Wnt3a and SHH activator induction, hHOs produce HOPX+ and PAX6+ progenitors and ZBTB20+PROX1+ dentate gyrus (DG) granule neurons. The transcriptomic signatures of hHOs reveal high similarity to the developing human hippocampus. We successfully detect neural activities from hHOs via the mMPC from this cyb-organoid. Compared with traditional planar devices, our non-invasive coupling offers an adaptor for recording neural signals from 3D models.

Epigenetic regulation by KDM5A mediates the effects of prenatal PM2.5 exposure on hippocampal development and synaptic integrity through the Shh signaling pathway

Prenatal environmental exposure could be an essential health risk factor associated with neurodevelopmental disorders in offspring. However, the exact mechanisms underlying the impact of prenatal PM2.5 exposure on offspring cognition remain unclear. In our recent study using a PM2.5 exposed pregnant mouse model, we observed significant synaptic dysfunction in the hippocampi of the offspring. Concurrently, the epigenetic regulator of KDM5A and the Shh signaling pathway exhibited decreased activities. Significantly, changes in hippocampal KDM5A and Shh levels directly correlated with PM2.5 exposure intensity. Subsequent experiments revealed a marked reduction in the expression of Shh signaling and related synaptic proteins when KDM5A was silenced in cells. Notably, the effects of KDM5A deficiency were reversed significantly with the supplementation of a Shh activator. Furthermore, our findings indicate that Shh activation significantly attenuates PM2.5-induced synaptic impairments in hippocampal neurons. We further demonstrated that EGR1, a transcriptional inhibitor, plays a direct role in KDM5A’s regulation of the Shh pathway under conditions of PM2.5 exposure. Our results suggest that the KDM5A’s inhibitory regulation on the Shh pathway through the EGR1 gene is a crucial epigenetic mechanism underlying the synaptic dysfunction in hippocampal neurons caused by maternal PM2.5 exposure. This emphasizes the role of epigenetic regulations in neurodevelopmental disorders caused by environmental factors.

Impact of Sonic Hedgehog-dependent sphenoid bone defect on craniofacial growth.

The main objective of this study was to evaluate how an apparently minor anomaly of the sphenoid bone, observed in a haploinsufficient mouse model for Sonic Hedgehog (Shh), affects the growth of the adult craniofacial region. This study aims to provide valuable information to orthodontists when making decisions regarding individuals carrying SHH mutation. The skulls of embryonic, juvenile and adult mice of two genotypes (Shh heterozygous and wild type) were examined and measured using landmark-based linear dimensions. Additionally, we analysed the clinical characteristics of a group of patients and their relatives with SHH gene mutations. In the viable Shh+/ - mouse model, bred on a C57BL/6J background, we noted the presence of a persistent foramen at the midline of the basisphenoid bone. This particular anomaly was attributed to the existence of an ectopic pituitary gland. We discovered that this anomaly led to premature closure of the intrasphenoidal synchondrosis and contributed to craniofacial deformities in adult mice, including a longitudinally shortened skull base. This developmental anomaly is reminiscent of that commonly observed in human holoprosencephaly, a disorder resulting from a deficiency in SHH activity. However, sphenoid morphogenesis is not currently monitored in individuals carrying SHH mutations. Haploinsufficiency of Shh leads to isolated craniofacial skeletal hypoplasia in adult mouse. This finding highlights the importance of radiographic monitoring of the skull base in all individuals with SHH gene mutations.

Sonic hedgehog restrains the ubiquitin-dependent degradation of SP1 to inhibit neuronal/glial senescence associated phenotypes in chemotherapy-induced peripheral neuropathy via the TRIM25-CXCL13 axis

IntroductionChemotherapy-induced peripheral neuropathy (CIPN) is a common complication that affects an increasing number of cancer survivors. However, the current treatment options for CIPN are limited. Paclitaxel (PTX) is a widely used chemotherapeutic drug that induces senescence in cancer cells. While previous studies have demonstrated that Sonic hedgehog (Shh) can counteract cellular dysfunction during aging, its role in CIPN remains unknown. ObjectivesHerein, the aim of this study was to investigate whether Shh activation could inhibits neuronal/glial senescence and alleviates CIPN. MethodsWe treated ND7/23 neuronal cells and RSC96 Schwann cells with two selective Shh activators (purmorphamine [PUR] and smoothened agonist [SAG]) in the presence of PTX. Additionally, we utilized a CIPN mouse model induced by PTX injection. To assess cellular senescence, we performed a senescence-associated β-galactosidase (SA-β-gal) assay, measured reactive oxygen species (ROS) levels, and examined the expression of P16, P21, and γH2AX. To understand the underlying mechanisms, we conducted ubiquitin assays, LC-MS/MS, H&E staining, and assessed protein expression through Western blotting and immunofluorescence staining. ResultsIn vitro, we observed that Shh activation significantly alleviated the senescence-related decline in multiple functions included SA-β-gal activity, expression of P16 and P21, cell viability, and ROS accumulation in DRG sensory neurons and Schwann cells after PTX exposure. Furthermore, our in vivo experiments demonstrated that Shh activation significantly reduced axonal degeneration, demyelination, and improved nerve conduction. Mechanistically, we discovered that PTX reduced the protein level of SP1, which was ubiquitinated by the E3 ligase TRIM25 at the lysine 694 (K694), leading to increased CXCL13 expression, and we found that Shh activation inhibited PTX-induced neuronal/glial senescence and CIPN through the TRIM25-SP1-CXCL13 axis. ConclusionThese findings provide evidence for the role of PTX-induced senescence in DRG sensory neurons and Schwann cells, suggesting that Shh could be a potential therapeutic target for CIPN.

Progenitor division and cell autonomous neurosecretion are required for rod photoreceptor sublaminar positioning

Migration is essential for the laminar stratification and connectivity of neurons in the central nervous system. In the retina, photoreceptors (PRs) migrate to positions according to birthdate, with early-born cells localizing to the basal-most side of the outer nuclear layer. It was proposed that apical progenitor mitoses physically drive these basal translocations non-cell autonomously, but direct evidence is lacking, and whether other mechanisms participate is unknown. Here, combining loss- or gain-of-function assays to manipulate cell cycle regulators (Sonic hedgehog, Cdkn1a/p21) with an in vivo lentiviral labelling strategy, we demonstrate that progenitor division is one of two forces driving basal translocation of rod soma. Indeed, replacing Shh activity rescues abnormal rod translocation in retinal explants. Unexpectedly, we show that rod differentiation also promotes rod soma translocation. While outer segment function or formation is dispensable, Crx and SNARE-dependent synaptic function are essential. Thus, both non-cell and cell autonomous mechanisms underpin PR soma sublaminar positioning in the mammalian retina.

MDB-25. LARGE CELL/ANAPLASTIC MEDULLOBLASTOMA REPRESENTS MORE THAN ONE DISEASE ENTITY

Abstract Medulloblastoma (MB) with predominant large cell or anaplastic cytological features have been identified to be associated to an aggressive clinical course and poor survival. Because cases can show mixed cytologies, these histologically defined MB types were merged in the recent WHO classification of tumors of the CNS to large cell/anaplastic (LC/A) MB. In this study, we collected FFPE samples of 137 LC/A MB of all age groups (0-39 years) diagnosed at the DGNN Brain Tumor Reference Center in Bonn, Germany, and performed a comprehensive histological, immunophenotypical, genetic and epigenetic analysis by next-generation panel sequencing (NGS), 450k/850k methylation bead-array hybridization and molecular inversion probe (MIP) technology. 73% of cases showed predominant anaplastic histology, 27% predominant large cell histology. Methylation-based subtyping and sequencing showed that the cohort of LC/A MB represented different biological entities: 6% WNT-MB, 7% SHH-MB-TP53-wildtype, 10% SHH-MB-TP53-mutant, and 77% non-WNT/non-SHH MB (with predominant epigenetic subtype II). High-resolution copy number profiling by MIP demonstrated MYC amplification in 40% of non-WNT/non-SHH MB. MYC amplification was highly associated to infant disease and predominant large cell histology. On the contrary, predominant anaplastic histology was highly associated to non-infant disease, MYCN amplification and SHH activation. By NGS we detected recurrent mutations in 37 MB-related genes; the frequency of mutations of individual MB-related genes differed significantly between the histologically and genetically defined MB types. Our data clearly demonstrate that large cell MB and anaplastic MB represent different biological entities. Therefore, and also in respect to the future development of targeted treatment approaches to these highly malignant MB types, they should be precisely diagnosed as separate MB entities.

PD36-02 A COMPOSITE BIOMARKER APPROACH TO SPARE NEOADJUVANT CHEMOTHERAPY IN SELECT MUSCLE-INVASIVE BLADDER CANCER PATIENTS

You have accessJournal of UrologyCME1 Apr 2023PD36-02 A COMPOSITE BIOMARKER APPROACH TO SPARE NEOADJUVANT CHEMOTHERAPY IN SELECT MUSCLE-INVASIVE BLADDER CANCER PATIENTS Joep De Jong, A. Gordon Robertson, Nick Beije, John Martens, Ellen Zwarthoff, Arno Van Leenders, Ewan Gibb, and Joost Boormans Joep De JongJoep De Jong More articles by this author , A. Gordon RobertsonA. Gordon Robertson More articles by this author , Nick BeijeNick Beije More articles by this author , John MartensJohn Martens More articles by this author , Ellen ZwarthoffEllen Zwarthoff More articles by this author , Arno Van LeendersArno Van Leenders More articles by this author , Ewan GibbEwan Gibb More articles by this author , and Joost BoormansJoost Boormans More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003334.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Clinical guidelines recommend neoadjuvant chemotherapy (NAC) for the treatment of muscle-invasive bladder cancer (MIBC). However, NAC prior to radical cystectomy (RC) is associated with treatment-related toxicity and confers a modest survival benefit. Here, we aimed to identify MIBC patients who may avoid NAC by combining circulating tumor cell status and molecular subtypes in a composite biomarker approach. METHODS: TURBT tissue specimen were collected from clinical stage T2-T4aN0-N1M0 MIBC cases that were included within the CirGuidance study (NL3954), a prospective trial that analyzed circulating tumor cell (CTC) status in a patient’s blood sample using the CELLSEARCH system. For the present study, transcriptome-wide expression profiling was performed on 234 TURBT samples using an array-based approach. Molecular subtypes, long non-coding RNA (lncRNA) based FGFR3+ status and gene signatures were determined as described previously (de Jong et al., Genome Med. 2019). The primary endpoint of this study was cancer-specific mortality (CSM), calculated as the date of study inclusion till date of bladder cancer related death. Median follow-up was 28.8 (IQR: 16.6-40.2) months. RESULTS: Of 234 patients, 21 (9%) were treated with NAC and RC, while 213 (91%) received RC alone. A CTC-negative status was observed in 172 (81%) of RC-only cases. Molecular subtyping identified 28 luminal FGFR3+ cases with high FGFR3, SHH and p53 pathway activity, and lower EMT hallmark scores. Adjusting for clinical risk factors, both CTC and FGFR3+ status were significant predictors for cancer specific mortality on MVA (p<0.05). Of interest, the subgroup of FGFR3+ cases that were CTC-negative (N=26) showed most favorable outcomes with only one CSM event after a median of 33.4 (IQR: 24.8-44.5) months of follow-up. CONCLUSIONS: Using a composite biomarker approach of blood-based CTC status and molecular subtyping, we identified a biologically distinct subgroup of MIBC with favorable prognosis after RC-only, validating the performance of a previously developed lncRNA based FGFR3+ classifier. Clinical trials which withhold NAC from CTC-negative, FGFR3+ MIBC patients are warranted. Source of Funding: Gene expression profiles were generated using the Decipher Bladder assay © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e980 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joep De Jong More articles by this author A. Gordon Robertson More articles by this author Nick Beije More articles by this author John Martens More articles by this author Ellen Zwarthoff More articles by this author Arno Van Leenders More articles by this author Ewan Gibb More articles by this author Joost Boormans More articles by this author Expand All Advertisement PDF downloadLoading ...

Interference of sympathetic overactivation restores limbal stem/progenitor cells function and accelerates corneal epithelial wound healing in diabetic mice

Diabetic keratopathy (DK), the diabetic complication in the cornea, is characterized by the delayed epithelial regeneration and sensory nerve degeneration. The involvement of limbal stem/progenitor cells (LSPCs) dysfunction has been reported, however the pathogenic mechanisms remain unclear. Here, we confirmed the dysfunction of LSPCs in diabetic mouse and human corneas. The sympathetic nerve in the cornea was adjacent to LSPCs, and the sympathetic overactivation was found in diabetic mice. Surgical and pharmacological ablation of sympathetic nerves rescued the LSPCs function and promoted corneal epithelial regeneration in diabetic mice. In contrast, both topical norepinephrine (NE) application and chemogenetic sympathetic overactivation directly impaired the stemness and proliferation characteristics of LSPCs, as well as the normal epithelial regeneration. Moreover, we identified that β2-adrenoceptor (Adrb2) was the predominant adrenergic receptor expressed in LSPCs by corneal limbal single-cell sequencing and real time PCR (RT-PCR) analysis of sorted LSPCs. The Adrb2 knockout mice exhibited the enhancement of epithelial regeneration and LSPCs function, compared with the wild-type mice. Similarly, topical application of the Adrb2 specific antagonist ICI 118, 551 effectively accelerated diabetic corneal epithelial regeneration with the restored LSPCs function. Mechanistically, sonic hedgehog (Shh) activity mediated the downstream effects of NE-Adrb2 signaling pathway in regulating LSPCs and epithelial regeneration. Taken together, our data revealed the involvement of sympathetic overactivation in the impairment of diabetic LSPCs function and corneal epithelial regeneration through the NE-Adrb2-Shh signaling pathway. The interference of sympathetic overactivation may provide novel treatment strategies for diabetic keratopathy.

Novel Approach to the Hedgehog Signaling Pathway: Combined Treatment of SMO and PTCH Inhibitors

Purpose: Abnormal Sonic Hedgehog signaling Pathway (Shh) activation is crucial for development of cancer stem cells, neoplastic growth and epithelial-mesenchymal transition processes in adulthood. Activation of Hedgehog signaling pathway may induces the changes in cilia found in the cell membrane, iniciates the Gli1 transcription factor that is translocated to the cell nucleus and finally, the target genes are transcribed. In this study, invastigation of the antiproliferative, anti-invasive and antimigrative effect of the combined use of robotnikinin (Ptch1 antagonist) and vismodegib (Smo inhibitor) on the hedgehog signaling pathway was aimed. &#x0D; &#x0D; Material and Methods: After demonstarting the presence of the hedgehog signaling pathway in the glioblastoma cell line U87-MG, the effect of the combined use of the robotnikinin and the vismodegib on the hedgehog signaling pathway was investigated. In-vitro cell proliferation, migration, and invasion analysis of the combination of antagonist and inhibitor and in silico drug-likeness analysis were performed.&#x0D; &#x0D; Results: Two different combinations of robotnikinin and vismodegib were tested. In vitro studies show that the combined use of agents in combined treatments of Smo and Ptch1is more effective than their individual usage.&#x0D; &#x0D; Conclusion: Inhibition of the hedgehog signaling pathway with specific inhibitors and antagonists is considered an innovative strategy for cancer therapy.

Evolutionary genetics of flipper forelimb and hindlimb loss from limb development-related genes in cetaceans

BackgroundCetacean hindlimbs were lost and their forelimb changed into flippers characterized by webbed digits and hyperphalangy, thus allowing them to adapt to a completely aquatic environment. However, the underlying molecular mechanism behind cetacean limb development remains poorly understood.ResultsIn the present study, we explored the evolution of 16 limb-related genes and their cis-regulatory elements in cetaceans and compared them with that of other mammals. TBX5, a forelimb specific expression gene, was identified to have been under accelerated evolution in the ancestral branches of cetaceans. In addition, 32 cetacean-specific changes were examined in the SHH signaling network (SHH, PTCH1, TBX5, BMPs and SMO), within which mutations could yield webbed digits or an additional phalange. These findings thus suggest that the SHH signaling network regulates cetacean flipper formation. By contrast, the regulatory activity of the SHH gene enhancer—ZRS in cetaceans—was significantly lower than in mice, which is consistent with the cessation of SHH gene expression in the hindlimb bud during cetacean embryonic development. It was suggested that the decreased SHH activity regulated by enhancer ZRS might be one of the reasons for hindlimb degeneration in cetaceans. Interestingly, a parallel / convergent site (D42G) and a rapidly evolving CNE were identified in marine mammals in FGF10 and GREM1, respectively, and shown to be essential to restrict limb bud size; this is molecular evidence explaining the convergence of flipper-forelimb and shortening or degeneration of hindlimbs in marine mammals.ConclusionsWe did evolutionary analyses of 16 limb-related genes and their cis-regulatory elements in cetaceans and compared them with those of other mammals to provide novel insights into the molecular basis of flipper forelimb and hindlimb loss in cetaceans.

28P SHH pathway-targeted therapy in a relapsed medulloblastoma

Recurrent medulloblastoma is a deleterious disease with a 5-year survival of less than 5% and no recognized standard treatment. In recent years, we have learned about the molecular characterization of the different types of medulloblastoma with four subgroups also appearing different subgroups, each with its oncogenic pathway activation. SHH activation is the hallmark of medulloblastoma group 2 and several molecules have been developed to block this signaling mechanism, including SMO inhibitors, that are approved for the treatment of basal cell carcinoma.

Mesenchymal FGFR1 and FGFR2 control patterning of the ureteric mesenchyme by balancing SHH and BMP4 signaling.

The coordinated development of the mesenchymal and epithelial progenitors of the murine ureter depends on a complex interplay of diverse signaling activities. We have recently shown that epithelial FGFR2 signaling regulates stratification and differentiation of the epithelial compartment by enhancing epithelial Shh expression, and mesenchymal SHH and BMP4 activity. Here, we show that FGFR1 and FGFR2 expression in the mesenchymal primordium impinges on the SHH/BMP4 signaling axis to regulate mesenchymal patterning and differentiation. Mouse embryos with conditional loss of Fgfr1 and Fgfr2 in the ureteric mesenchyme exhibited reduced mesenchymal proliferation and prematurely activated lamina propria formation at the expense of the smooth muscle cell program. They also manifested hydroureter at birth. Molecular profiling detected increased SHH, WNT and retinoic acid signaling, whereas BMP4 signaling in the mesenchyme was reduced. Pharmacological activation of SHH signaling in combination with inhibition of BMP4 signaling recapitulated the cellular changes in explant cultures of wild-type ureters. Additional experiments suggest that mesenchymal FGFR1 and FGFR2 act as a sink for FGF ligands to dampen activation of Shh and BMP receptor gene expression by epithelial FGFR2 signaling.