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Failure of myelin regeneration by oligodendrocytes contributes to progressive decline in many neurological diseases. Here, using in vitro and in vivo rodent models, functional blockade, and mouse brain demyelination, we demonstrate that Sonic hedgehog (Shh) expression in a subset of oligodendrocyte progenitor cells precedes the expression of myelin basic protein (MBP), a major myelin sheath protein. Primary cultures of rodent cortical oligodendrocytes show that Shh mRNA and protein are upregulated during oligodendrocyte maturation before the upregulation of MBP expression. Importantly, almost all MBP-positive cells are Shh positive during differentiation. During remyelination, we identify a rapid induction of Shh mRNA and peptide in oligodendroglial cells present in the demyelinated corpus callosum of mice, including a population of PDGFRα-expressing cells. Shh invalidation by an adeno-associated virus strategy demonstrates that the downregulation of Shh impairs the differentiation of oligodendrocytes in vitro and decreases MBP and myelin proteolipid protein expression in the demyelinated mouse brain at late stages of remyelination. We also report a parallel expression of Shh and MBP in oligodendroglial cells during early post-natal myelination of the mouse brain. Thus, we identify a crucial Shh signal involved in oligodendroglial cell differentiation and remyelination, with potential interest in the design of better-targeted remyelinating therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background Objectives Methods Results Conclusions Basal cell carcinomas (BCCs) are the most common cancers worldwide. Although most BCCs are amenable to local treatment, there are limited therapeutic options for surgically unresectable locally advanced and metastatic BCCs. Activation of the sonic hedgehog signalling pathway plays a significant role in the development of most BCCs. Hedgehog pathway inhibitors (HPIs) can be used to inhibit this pathway. Efficacy and safety data on HPI use in Australia is scarce.This study aims to present the effectiveness and safety of HPI at a tertiary dermatology referral centre.We conducted a retrospective analysis of the clinical charts of all patients with BCC treated with an HPI at a tertiary Dermatology referral centre in New South Wales, Australia from 1 January 2016 to 1 July 2023.Twenty‐three patients with BCCs were treated with an HPI; 11 locally advanced, 8 multiple, 3 basal cell naevus syndrome and 1 metastatic. All patients were of Caucasian background, with a median age of 56. Across 41 treatment cycles, the median treatment duration was 4 months. The overall response rate (ORR) was 20/23 (87%) and complete response (CR) rate was 9/23 (39%); patients treated with sonidegib achieved an ORR of 11/12 (92%) and CR of 4/12 (33%), and vismodegib‐treated patients achieved an ORR of 9/11 (82%) and CR of 5/11 (45%). Patients who responded to HPI treatment also responded to a subsequent HPI rechallenge. Common treatment emergent adverse events (TEAEs) included muscle spasms, dysgeusia and alopecia. Dysgeusia was more frequent with vismodegib than sonidegib (p = 0.0001). There was no evidence to suggest a difference in other TEAEs between the two HPIs. Four treatment cycles were stopped due to grade 3 muscle spasm.In our cohort of 23 patients being treated with HPI, the ORR was 87% and CR was 39%. All patients who experienced TEAEs and had a drug holiday successfully responded to HPI rechallenge. TEAEs, particularly muscle spasms, are common reasons for treatment cessation. Clinicians should implement strategies to mitigate TEAE to improve drug survivability. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is the most severe bone tumor in children. A chemotherapy regimen includes a combination of high-dose Methotrexate (MTX), doxorubicin, and cisplatin. These drugs cause acute and chronic side effects, such as infections, thrombocytopenia, neutropenia, DNA damage, and inflammation. Therefore, to identify new therapeutic strategies, effective and with a safety profile, is necessary. The Hedgehog (Hh) signaling pathway involved in tumorigenesis is active in OS. Hh components Patched receptor 1 (PTCH1), Smoothened (SMO), and glioma-associated oncogene homolog transcription factors (GLI1 and GLI2) are overexpressed in OS cell lines and patient samples. Curcumin (CUR)—with antioxidant and anti-cancer properties—downregulates Hh components in cancer, inhibiting progression. This study investigates CUR effects on the MG-63 OS cell line, alone and combined with MTX, to propose a novel therapeutic approach. Our study suggests CUR as a novel therapeutic agent in OS, particularly when combined with MTX. Targeting the Hh signaling pathway, CUR and MTX showed significant pro-apoptotic effects, increasing the BAX/Bcl-2 ratio and total apoptotic cell percentage. They reduced the expression of Hh pathway components (PTCH1, SMO, GLI1, and GLI2), inhibiting OS cell proliferation, survival, and invasion. CUR and MTX combined determined a β-Catenin decrease and a trend toward reducing NF-kB and matrix metalloproteinases (MMP-2 and MMP-9). Our findings suggest CUR as a support to OS treatment, improving outcomes and reducing the adverse effects of current therapies. [ABSTRACT FROM AUTHOR]

Protein phosphatase 2A (PP2A), one of the most abundant protein phosphatases, has divergent functions in multiple types of cells. Its inactivation has been closely associated with leukemia diseases. However, the physiological function of PP2A for hematopoiesis has been poorly understood in organisms. Drosophila hematopoiesis parallels the vertebrate counterpart in developmental and functional features but involves a much simpler hematopoietic system. Here, utilizing the Drosophila major larval hematopoietic organ lymph gland, we studied the function of PP2A for hematopoiesis in vivo. By knocking down the expression of Pp2A‐29B that encodes the scaffold subunit of the PP2A holoenzyme complex, we found that PP2A silencing in the differentiating hemocytes resulted in their excessive proliferation. Furthermore, this PP2A inhibition downregulated the expression of Smoothened (Smo), a crucial component in the Hedgehog pathway, and smo overexpression was able to rescue the phenotypes of PP2A depletion, indicating that Smo functions as a downstream effector of PP2A to restrict the hemocyte proliferation. PDGF/VEGF‐receptor (Pvr) overexpression also restored the Smo expression and lymph gland morphology of PP2A silencing, suggesting a PP2A‐Pvr‐Smo axis to regulate lymph gland growth and hemocyte proliferation. Moreover, inhibiting PP2A activity in the blood progenitor cells promoted their differentiation, but which was independent with Smo. Together, our data suggested that PP2A plays a dual role in the Drosophila lymph gland by preserving the progenitor population and restraining the hemocyte proliferation, to properly regulate the hematopoietic process. [ABSTRACT FROM AUTHOR]

The drug resistance challenges in acute myeloid leukemia have been linked the hedgehog (HH) signaling pathway activation by many studies. Furthermore, studies have shown that the components of the HH pathway, including 12‐pass transmembrane receptor Patched (PTCH), the 7‐pass transmembrane signal transduction protein Smoothened (SMO), and all three GLI transcription factors, are expressed in AML. Thus, pharmacological inhibition of SMO in the HH signaling in these cells has anti‐leukemic effects. However, the first SMO inhibitor specific to AML (Glasdegib) has been approved based on improved overall survival rates, but alternative approaches are still required. Thus we employed druglikeness screening, molecular docking, molecular dynamics simulation, non‐equilibrium free energy and in silico pharmacokinetics profiling on bioactive compounds from Vernonia amygdalina against SMO protein target. From 30 bioactive compounds in V. amygdalina, 13 had drug‐like potential, 5 had binding energy between −9.2 to −9.9 kcal/mol. Cryptolepine, neocryptolepine and isocryptolepine with the least BE showed good stability, dynamics over 300 ns of MD run and higher binding affinities via the Jarzynski's identity. Hence, these bioactive compounds may serve as potential drugs for the treatment of acute myeloid leukemia by targeting the SMO protein after wet lab experiments. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC) is a malignant disease worldwide. Angiopoietin-like protein 4 (ANGPTL4) plays a role in pathophysiological processes, including metabolic reprogramming, angiogenesis, proliferation, and metastasis. Current evidence shows conflicting findings regarding the role of ANGPTL4 in the progression of GC. ANGPTL4 in GC was confirmed through bioinformatic analysis and immunofluorescence staining. The impact of ANGPTL4 was subsequently validated in GC cell lines using various assays, including 5-ethynyl-2-deoxyuridine (EdU), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Flow Cytometry (FCM), wound healing, transwell, tube formation, chorioallantoic membrane model, and nude mouse model assays. RNA-seq analysis, polymerase chain reaction (PCR), western blotting (WB), immunofluorescence (IF) and coimmunoprecipitation (co-IP) were conducted to determine the potential downstream mechanism of ANGPTL4. In SNU5 and MKN7 cells, ANGPTL4 was found to augment proliferation, migration, invasion, evasion of apoptosis, and angiogenesis. Conversely, in the AGS cell line, ANGPTL4 was observed to suppress these processes. Notably, the overexpression of ANGPTL4 in AGS cells led to the upregulation of LGALS7, which has emerged as a pivotal factor contributing to the manifestation of an anticancer phenotype induced by ANGPTL4. LGALS7, which is involved in the regulation of the hedgehog pathway and subsequent promotion of GC progression through various processes, such as proliferation, migration, apoptosis evasion, angiogenesis, and lymphangiogenesis, was found to contribute to the contradictory effects of ANGPTL4. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated that ablative fractional laser (AFL) can inhibit the hedgehog pathway, enhance immune infiltration and clear basal cell carcinomas (BCCs) in murine models. In this study, we applied RNA sequencing to further characterise the impact of AFL on the transcriptome of murine skin containing early‐stage microscopic BCCs, contrasting it with the effects of topical application of the hedgehog inhibitor vismodegib. Our results showed that BCC induction in murine skin was primarily linked to gene upregulation (significantly upregulated genes: 277, significantly downregulated genes: 24). Characterisation of these genes with Ingenuity Pathway Analysis showed that tumour induction was associated with activation of BCC and Sonic Hedgehog signalling. Both AFL and vismodegib treatments reversed these changes, with vismodegib demonstrating superior performance by reversing most of the upregulated genes (AFL: 59/277; vismodegib: 180/277). Surprisingly, Ingenuity Pathway Analysis also revealed that both AFL and vismodegib treatments caused considerable immune cell infiltration. Based on gene set enrichment analysis and cell type deconvolution, AFL treatment resulted in the largest immune cell recruitment, which for both treatments primarily consisted of infiltrating neutrophils, macrophages and monocytes. In conclusion, the distinct effects observed in BCC skin following AFL and vismodegib treatment suggest key differences between the two interventions. Future applications of AFL or vismodegib treatments could leverage their individual effects, for example by combining the effect of AFL on the immune system with other topical treatments. [ABSTRACT FROM AUTHOR]

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing the primary cilium, the cell's antenna, which acts as a signaling hub. Fuz, an effector of planar cell polarity signaling, regulates Shh signaling by facilitating cilia formation, and the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of the genes encoding these proteins is similar; however, their functional relationship has not been previously explored. This study identified the genetic and biochemical linkage between Fuz and Gpr161 in mouse neural tube development. Fuz was found to be genetically epistatic to Gpr161 with respect to regulation of Shh signaling in mouse neural tube development. The Fuz protein biochemically interacts with Gpr161, and Fuz regulates Gpr161-mediated ciliary localization, a process that might utilize β-arrestin 2. Our study characterizes a previously unappreciated Gpr161-Fuz axis that regulates Shh signaling during mouse neural tube development. [ABSTRACT FROM AUTHOR]

Basal cell carcinoma (BCC) is the most frequent of all cancers, with an increasing incidence. The first line therapy is surgical excision, but topical therapies can be used in low-risk superficial BCCs, while the more advanced, unresectable, or metastatic BCCs benefit from systemic therapies with hedgehog inhibitors and immunotherapy. The purpose of this review is to highlight local and systemic immunotherapies and their efficacy in the management of BCCs. Local therapies can be considered in superficial and low-risk nodular BCCs, with imiquimod frequently used for its antitumor and immunoregulatory properties. Imiquimod alone demonstrated higher histological clearance rates, but patients treated with imiquimod experienced more adverse events than ones treated with other therapies. Imiquimod can be used as an adjuvant before Mohs micrographic surgery and can also be combined with other local therapies, like curettage, electrodesiccation, cryosurgery, and photodynamic therapy, with some treatment methods yielding results comparable with the surgery. Interferons and Interleukin-2 were evaluated in a small number of studies with different results. Systemic immunotherapies with programmed death-ligand 1 (PD-L1) inhibitors showed inconsistent results in patients with advanced BCCs, being effective in some patients that progressed on or were intolerant to hedgehog pathway inhibitors (HHI). [ABSTRACT FROM AUTHOR]

Endometrial Cancer (EC) is one of the most common gynecological malignancies. Despite its prevalence, molecular pathways, such as the Sonic Hedgehog (SHH) pathway, have not been extensively studied in the context of EC. This study aims to explore the clinical implications of SHH expression in EC, potentially uncovering new insights into the disease's pathogenesis and offering valuable insights for therapeutic strategies in EC. We utilized data from The Cancer Genome Atlas (TCGA) to divide the dataset into 'High SHH' and 'Low SHH' groups based on a gene signature score derived from SHH pathway-related genes. We explored the clinical and tumor characteristics of these groups, focusing on key cancer hallmarks, including stemness, proliferation, cytolytic activity, tumor micro-environment, and genomic instability. 'High SHH' tumors emerged as a distinct category with favorable clinical and molecular features. These tumors exhibited lower proliferation rates, reduced angiogenesis, and diminished genomic instability, indicating a controlled and less aggressive tumor growth pattern. Moreover, 'High SHH' tumors displayed lower stemness, highlighting a less invasive phenotype. The immune micro-environment in 'High SHH' tumors was enriched with immune cell types, such as macrophage M0, monocytes, B cells, CD8 T cells, CD4 T cells, follicular helper T cells, and natural killer cells. This immune enrichment, coupled with higher cytolytic activity, suggested an improved anti-tumor immune response. Our study sheds light on the clinical significance of Sonic signaling in EC. 'High SHH' tumors exhibit a unique molecular and clinical profile associated with favorable cancer hallmarks, lower grades, and better survival. These findings underscore the potential utility of SHH expression as a robust prognostic biomarker, offering valuable insights for tailored therapeutic strategies in EC. Understanding the SHH pathway's role in EC contributes to our growing knowledge of this cancer and may pave the way for more effective treatment strategies in the future. [ABSTRACT FROM AUTHOR]

The burden of senescent hepatocytes correlates with the severity of metabolic dysfunction–associated steatotic liver disease (MASLD), but the mechanisms driving senescence and how it exacerbates MASLD are poorly understood. Hepatocytes experience lipotoxicity and become senescent when Smoothened (Smo) is deleted to disrupt Hedgehog signaling. We aimed to determine whether the secretomes of Smo-deficient hepatocytes perpetuate senescence to drive MASLD progression. RNA- Seq analysis of liver samples from human and murine cohorts with MASLD confirmed that hepatocyte populations in MASLD livers were depleted of Smo+ cells and enriched with senescent cells. When fed a choline-deficient, amino acid–restricted high-fat diet (CDA-HFD) to induce MASLD, Smo– mice had lower antioxidant markers and developed worse DNA damage, senescence, steatohepatitis, and fibrosis than did Smo+ mice. Sera and hepatocyte-conditioned medium from Smo– mice were depleted of thymidine phosphorylase (TP), a protein that maintains mitochondrial fitness. Treating Smo– hepatocytes with TP reduced senescence and lipotoxicity, whereas inhibiting TP in Smo+ hepatocytes had the opposite effect and exacerbated hepatocyte senescence, steatohepatitis, and fibrosis in CDA-HFD–fed mice. We conclude that inhibition of Hedgehog signaling in hepatocytes promoted MASLD by suppressing hepatocyte production of proteins that prevent lipotoxicity and senescence. [ABSTRACT FROM AUTHOR]

Cyclopamine, a natural alkaloid, can act as an agonist when it binds to the Cysteine-Rich Domain (CRD) of Smoothened receptor and as an antagonist when it binds to the Transmembrane Domain (TMD). To study the effect of cyclopamine binding to each site experimentally, mutations in the other site are required. Hence, simulations are critical for understanding the WT activity due to binding at different sites. Using multi-milliseconds long aggregate MD simulations combined with Markov state models and machine learning, we explore the dynamic behavior of cyclopamine's interactions with different domains of WT SMO. A higher population of the active state at equilibrium, a lower free energy barrier of ~2 kcal/mol, and expansion of hydrophobic tunnel to facilitate cholesterol transport agrees with cyclopamine's agonistic behavior when bound to CRD. A higher population of the inactive state at equilibrium, a higher free energy barrier of ~4 kcal/mol and restricted hydrophobic tunnel shows cyclopamine's antagonistic behavior when bound to TMD. With cyclopamine bound to both sites, there is a slightly larger inactive population at equilibrium and an increased free energy barrier (~3.5 kcal/mol) exhibiting an overall weak antagonistic effect. These findings show cyclopamine's domain-specific modulation of SMO regulates Hedgehog signaling and cholesterol transport. Millisecond scale MD simulations explain how cyclopamine modulates human Smoothened receptor activity based on different binding positions. [ABSTRACT FROM AUTHOR]

Liver fibrosis is characterized by excess reactive oxygen species (ROS) production, hepatic stellate cell (HSC) activation, and subsequent extracellular matrix deposition. Since complex pathways regulate HSC activation, the single‐drug therapy efficacy for live fibrosis often falls short of expectations. To address this issue, an HSC‐targeted multifunctional nanoparticle (NP) delivery system co‐loaded with cyclopamine (Cyc; hedgehog inhibitor) and bilirubin (BR; ROS‐scavenger) is designed. The NP, termed RHB, is constructed via chemically conjugating hydrophobic bilirubin to hyaluronic acid (HA), followed by inserting into a cRGDyK peptide modified‐PEG shell. RHB can effectively target activated HSCs in the fibrotic liver by recognizing of the cRGDyK peptide and HA with integrin αvβ3 and CD44 due to their high expression on HSCs. During liver fibrosis, RHB NPs intelligently released Cyc in response to elevated ROS, inhibiting hedgehog signaling and subsequent HSC activation. Meanwhile, RHB NPs exhibited ROS scavenging capability and activated nuclear factor erythroid 2‐related factor 2 (Nrf2) signaling in stimulated HSCs, deactivating HSC. Cyc‐loaded RHB NPs significantly reversed the inflammatory and fibrotic phenotypes in liver fibrotic mice without evident toxicity. In summary, the multifunctional NPs dual‐targeted activated HSCs and deactivated HSC via multiple signaling, effectively ameliorating liver fibrosis, showing great promise for hepatic fibrosis treatment. [ABSTRACT FROM AUTHOR]

Epithelial‐stromal interplay through chemomechanical cues from cells and matrix propels cancer progression. Elevated tissue stiffness in potentially malignant tissues suggests a link between matrix stiffness and enhanced tumor growth. In this study, employing chronic oral/esophageal injury and cancer models, it is demonstrated that epithelial–stromal interplay through matrix stiffness and Hedgehog (Hh) signaling is key in compounding cancer development. Epithelial cells actively interact with fibroblasts, exchanging mechanoresponsive signals during the precancerous stage. Specifically, epithelial cells release Sonic Hh, activating fibroblasts to produce matrix proteins and remodeling enzymes, resulting in tissue stiffening. Subsequently, basal epithelial cells adjacent to the stiffened tissue become proliferative and undergo epithelial‐to‐mesenchymal transition, acquiring migratory and invasive properties, thereby promoting invasive tumor growth. Notably, transcriptomic programs of oncogenic GLI2, mechano‐activated by actin cytoskeletal tension, govern this process, elucidating the crucial role of non‐canonical GLI2 activation in orchestrating the proliferation and mesenchymal transition of epithelial cells. Furthermore, pharmacological intervention targeting tissue stiffening proves highly effective in slowing cancer progression. These findings underscore the impact of epithelial‐stromal interplay through chemo‐mechanical (Hh‐stiffness) signaling in cancer development, and suggest that targeting tissue stiffness holds promise as a strategy to disrupt chemo‐mechanical feedback, enabling effective cancer treatment. [ABSTRACT FROM AUTHOR]

Animals use a small number of morphogens to pattern tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. Here, we used single-molecule imaging in reconstituted morphogen gradients and in tissue explants to determine that Hedgehog diffused extracellularly as a monomer, and rapidly transitioned between membrane-confined and -unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion. Instead, we developed a topology-limited diffusion model in which cell-cell gaps create diffusion barriers, which morphogens can only overcome by passing through a membrane-unconfined state. Under this model, SCUBE1 promoted Hedgehog secretion and diffusion by allowing it to transiently overcome diffusion barriers. This multiscale understanding of morphogen gradient formation unified prior models and identified knobs that nature can use to tune morphogen gradient sizes across tissues and organisms. [ABSTRACT FROM AUTHOR]

We have previously demonstrated a rapid secretion of matrix metalloproteinase‐2 (MMP‐2) in the ischemic brain. Since Scube2 can interact with Sonic hedgehog (Shh) to maintain blood–brain barrier (BBB) integrity via regulating the interaction between brain capillary endothelial cells (ECs) and perivascular astrocytes, and it is also a substrate of MMP‐2, we hypothesized that the secreted MMP‐2 could degrade Scube2 and contribute to ischemic BBB disruption. Using an in vitro ischemic model of 90‐min oxygen–glucose deprivation/3‐h reoxygenation (OGD/R) and an in vivo mouse stroke model of 90‐min middle cerebral artery occlusion (MCAO) with 3‐h reperfusion, we established an important role of MMP‐2‐mediated Scube2 degradation in early ischemic BBB disruption. Exposure of C8‐D1A cells and bEnd.3 cells to OGD/R increased MMP secretion in both cells, and C8‐D1A cells appeared to secrete more MMPs than bEnd.3 cells. Co‐IP and double‐immunostaining revealed that Scube2 co‐localized well with MMP‐2 in C8‐D1A cells and could be pulled down by MMP‐2 antibodies. In MCAO mice, Scube2 protein showed a drastic reduction in ischemic brain tissue, which was accompanied by suppressed expression of Shh and its downstream molecules. Of note, specific knockdown of astrocytic Scube2 with AAV‐shScube2 augmented MCAO‐induced Shh suppression and exacerbated BBB leakage and inflammatory reactions in the ischemic brain. Last, incubation of bEnd.3 cells with conditioned medium derived from OGD‐treated C8‐D1A cells led to a significant inhibition of the Shh pathway in bEnd.3 cells and degradation of VE‐cadherin and ZO‐1. Inhibition of MMP‐2 with SB‐3CT or over‐expression of Scube2 with plasmids in C8‐D1A cells alleviated the above effect of C8‐D1A cells‐derived conditioned medium. Taken together, our data indicate that ischemia‐induced secretion of MMP‐2 may contribute to early BBB disruption in ischemic stroke via interrupting the shared Scube2‐Shh pathway between brain capillary ECs and perivascular astrocytes. [ABSTRACT FROM AUTHOR]

Background: Positron emission tomography/computed tomography (PET/CT) with 18F-fluorodeoxyglucose (18F-FDG) is a firmly established tool in oncology and is gaining importance in dermato-oncology. However, its use in advanced basal cell carcinoma (BCC) is limited, with only a few case reports and a single study focused on vismodegib. This study evaluates the role of 18F-FDG PET/CT in advanced BCC treated with sonidegib. Methods: We retrospectively assessed the clinical data of patients with advanced BCC who underwent 18F-FDG PET/CT between January 2022 and January 2024. Inclusion criteria included histologically confirmed BCC, FDG-avid lesions on baseline PET/CT, and a minimum follow-up of 6 months. Metabolic response was assessed using the PET Response Criteria in Solid Tumors (PERCIST). Results: Four patients with advanced BCC treated with sonidegib were included, presenting with a total of 10 hypermetabolic lesions at baseline PET/CT. The mean interval between baseline and follow-up scans was 8.7 ± 1.6 months. According to PERCIST, two patients achieved a complete metabolic response (CMR), while the other two had stable metabolic disease (SMD). Low baseline-standardized uptake values (i.e., SUVmax, SUVmean) and reduced total lesion glycolysis (TLG) were associated with CMR. No relapses were observed during follow-up. Conclusions: This study suggests that 18F-FDG PET/CT may help identify advanced BCC patients who are likely to benefit from sonidegib treatment. Further research is needed to fully explore the potential of PET/CT in this specific clinical context. [ABSTRACT FROM AUTHOR]

Many cellular processes are regulated by proteasome-mediated protein degradation, including regulation of signaling pathways and gene expression. Among the pathways regulated by the ubiquitin–proteasome system is the Hedgehog pathway and its downstream effectors, the Gli transcription factors. Here we provide evidence that proteasomal activity is necessary for maintaining the activation of the Hedgehog pathway, and this crucial event takes place at the level of Gli proteins. We undertook extensive work to demonstrate the specificity of the observed phenomenon by ruling out the involvement of primary cilium, impaired nuclear import, failed dissociation from Sufu, microtubule stabilization, and stabilization of Gli repressor forms. Moreover, we showed that proteasomal-inhibition-mediated Hedgehog pathway downregulation is not restricted to the NIH-3T3 cell line. We demonstrated, using CRISPR/Ca9 mutagenesis, that neither Gli1, Gli2, nor Gli3 are solely responsible for the Hedgehog pathway downregulation upon proteasome inhibitor treatment, and that Cul3 KO renders the same phenotype. Finally, we report two novel E3 ubiquitin ligases, Btbd9 and Kctd3, known Cul3 interactors, as positive Hedgehog pathway regulators. Our data pave the way for a better understanding of the regulation of gene expression and the Hedgehog signaling pathway. [ABSTRACT FROM AUTHOR]

Uterine leiomyosarcoma (uLMS) is the most common type of uterine sarcoma, associated with poor prognosis, high rates of recurrence, and metastasis. Currently, the molecular mechanism of the origin and development of uLMS is limited. Bromodomain and extra-terminal (BET) proteins are involved in both physiological and pathological events. However, the role of BET proteins in the pathogenesis of uLMS is unknown. Here, we show for the first time that BET protein family members, BRD2, BRD3, and BRD4, are aberrantly overexpressed in uLMS tissues compared to the myometrium, with a significant change by histochemical scoring assessment. Furthermore, inhibiting BET proteins with their small, potent inhibitors (JQ1 and I-BET 762) significantly inhibited the uLMS proliferation dose-dependently via cell cycle arrest. Notably, RNA-sequencing analysis revealed that the inhibition of BET proteins with JQ1 and I-BET 762 altered several critical pathways, including the hedgehog pathway, EMT, and transcription factor-driven pathways in uLMS. In addition, the targeted inhibition of BET proteins altered several other epigenetic regulators, including DNA methylases, histone modification, and m6A regulators. The connections between BET proteins and crucial biological pathways provide a fundamental structure to better understand uterine diseases, particularly uLMS pathogenesis. Accordingly, targeting the vulnerable epigenome may provide an additional regulatory mechanism for uterine cancer treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: Basal cell carcinoma (BCC) is the most common form of skin cancer. In advanced stage, BCC exhibits aggressive behavior, leading to local tissue destruction. Thus, a multidisciplinary approach and specialized treatment strategies are required for effective disease management and improvement of the patient quality of life. For low-risk disease, surgical intervention is the modality of choice while radiotherapy is indicated in unresectable tumor, positive margins post-surgery when re-excision is not feasible, and in the presence of high-risk features for recurrence. For locally advanced and metastatic disease, the emergence of systemic treatments including hedgehog inhibitors and immune checkpoint inhibitors has significantly improved the outcomes. Nonetheless, developing resistance to therapy presents a significant challenge in management. Consequently, development of novel therapeutic strategies is ongoing to address improving treatment efficacy. Basal cell carcinoma (BCC) is the most common type of cancer with an estimated 3.6 million cases diagnosed annually in the US alone. While most cases are treatable with low recurrence rates, 1–10% progress to an advanced stage which can behave aggressively, leading to local destruction and posing substantial challenges in management. The pathogenesis often involves dysregulation of the patched/hedgehog protein family, a pivotal pathway targeted by recently approved therapies. Furthermore, the role of immunotherapy is evolving in this type of tumor as we learn more about tumor microenvironment dynamics. In recent years, there have been advancements in the therapeutic landscape of advanced BCC, offering patients new hope and options for managing this complex and potentially life-threatening condition. In this review, we aim to provide a comprehensive overview of this disease, including the risk factors, underlying pathogenesis, current treatment options of advanced disease, and the ongoing exploration and development of novel therapies. [ABSTRACT FROM AUTHOR]

Hedgehog pathway inhibitors (HHIs) have broadened the treatment options available for patients with advanced basal cell carcinoma (BCC) for whom traditional therapeutic approaches are not feasible or effective. Sonidegib and vismodegib are oral HHIs that were approved for treatment of patients with advanced BCC after demonstrating promising efficacy in the pivotal Phase II BOLT (NCT01327053) and ERIVANCE (NCT00833417) trials, respectively. However, the incidence and types of treatment-emergent adverse events (AEs) observed with these agents may limit continuous use of HHIs and ultimately impact clinical outcomes. In this review, we summarize the safety and tolerability profiles of sonidegib and vismodegib and discuss potential management strategies for HHI class-effect AEs, including muscle spasms, creatine phosphokinase increase, alopecia, and dysgeusia. These AEs primarily occur early in treatment and can lead to treatment discontinuation. Differences in the pharmacokinetic profiles of sonidegib and vismodegib may contribute to the variability noted in times to onset and resolution of these and other AEs. Evidence suggests that protocol modifications, such as treatment interruptions and dose reductions, are effective ways to manage AEs while maintaining disease control. Nonpharmacologic and pharmacologic interventions may also be considered as part of an AE management strategy. Overall, healthcare providers and patients with advanced BCC should be aware of the HHI class-effect AEs and plan effective management strategies to avoid treatment discontinuation and optimize therapeutic response. [ABSTRACT FROM AUTHOR]

HER2-positive breast cancer, representing 15–20% of all breast cancer cases, often develops resistance to the HER2-targeted therapy trastuzumab. Unfortunately, effective treatments for advanced HER2-positive breast cancer remain scarce. This study aims to investigate the roles of ITGβ3, and Hedgehog signaling in trastuzumab resistance and explore the potential of combining trastuzumab with cilengitide as a therapeutic strategy. Quantitative gene expression analysis was performed to assess the transcription of EMT (epithelial–mesenchymal transition) markers Slug, Snail, Twist2, and Zeb1 in trastuzumab-resistant HER2-positive breast cancer cells. The effects of ITGβ3 and Hedgehog signaling were investigated. Additionally, the combination therapy of trastuzumab and cilengitide was evaluated. Acquired trastuzumab resistance induced the transcription of Slug, Snail, Twist2, and Zeb1, indicating increased EMT. This increased EMT was mediated by ITGB3 and Hedgehog signaling. ITGβ3 regulated both the Hedgehog pathway and EMT, with the latter being independent of the Hedgehog pathway. The combination of trastuzumab and cilengitide showed a synergistic effect, reducing both EMT and Hedgehog pathway activity. Targeting ITGβ3 with cilengitide, combined with trastuzumab, effectively suppresses the Hedgehog pathway and EMT, offering a potential strategy to overcome trastuzumab resistance and improve outcomes for HER2-positive breast cancer patients. [ABSTRACT FROM AUTHOR]

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development, including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and include regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth. [ABSTRACT FROM AUTHOR]

Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson's and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson's. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's. [ABSTRACT FROM AUTHOR]

Phenotypic assays detect small‐molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule‐target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4‐g]quinoline (PQ) pseudo‐natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA‐box binding protein‐associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains. [ABSTRACT FROM AUTHOR]

In Caenorhabditis elegans , expanded families of divergent Hedgehog-related and patched-related proteins promote numerous processes ranging from epithelial and sense organ development to pathogen responses to cuticle shedding during the molt cycle. The molecular functions of these proteins have been mysterious since nematodes lack a canonical Hedgehog signaling pathway. Here we show that Hedgehog-related proteins are components of the cuticle and precuticle apical extracellular matrices that coat, shape, and protect external epithelia. Of four Hedgehog-related proteins imaged, two (GRL-2 and GRL-18) stably associated with the cuticles of specific tubes and two (GRL-7 and WRT-10) labeled precuticle substructures such as furrows or alae. We found that wrt-10 mutations disrupt cuticle alae ridges, consistent with a structural role in matrix organization. We hypothesize that most nematode Hedgehog-related proteins are apical extracellular matrix components, a model that could explain many of the reported functions for this family. These results highlight ancient connections between Hedgehog proteins and the extracellular matrix and suggest that any signaling roles of C. elegans Hedgehog-related proteins will be intimately related to their matrix association. [ABSTRACT FROM AUTHOR]

Aims: Current evidence suggests that there is no completely effective method for endometriosis (EMS) without trauma due to diverse adverse effects. Reliable evidence illustrates that inhibiting ferroptosis is a potential strategy for EMS. We sufficiently verified that the expression of endogenous protein PDZ and LIM domain 3 (PDLIM3) was significantly increased in EMS. Methods: PDLIM3 knockdown reduced primary ectopic endometrial stromal cells' (EESCs) viability and migration, and elevated ferroptosis signaling indicators including Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS) in EESCs. Results: Mechanistic studies revealed that inhibition of PDLIM3 accelerated glioma-associated oncogene-1 (Gli1) degradation and further deactivated Hedgehog signaling. Gli1 inhibitor, GANT61, abrogated the impact of PDLIM3 deletion on EESC growth, migration, and ferroptosis. In vivo experiments suggested that PDLIM3 reduction repressed the growth of endometrial lesions. Likewise, repression of PDLIM3 promoted ferroptosis and attenuated Hedgehog signaling in endometrial lesions. Conclusions: Collectively, silencing of PDLIM3 facilitates ferroptosis in EMS by inducing Gli1 degradation and blocking Hedgehog signaling. It may provide an alternative strategy for developing therapeutic agents of EMS in the future. [ABSTRACT FROM AUTHOR]

Purpose: The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth. Methods: Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting. Results: Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth. Conclusion: Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease. [ABSTRACT FROM AUTHOR]

Understanding the developmental processes and signaling pathways involved in larval myogenesis and metamorphosis is crucial for comprehending the life history and adaptive strategies of marine organisms. In this study, we investigated the temporal and spatial patterns of myogenesis in the mussel Mytilus coruscus (Mc), focusing on the emergence and transformation of major muscle groups during different larval stages. We also explored the role of the Hedgehog (Hh) signaling pathway in regulating myogenesis and larval metamorphosis. The results revealed distinct developmental stages characterized by the emergence of specific muscular components, such as velum retractor muscles and anterior adductor muscles, in D-veliger and umbo larvae, which are responsible for the planktonic stage. In the pediveliger stage, posterior ventral, posterior adductor, and foot muscles appeared. After larval metamorphosis, the velum structure and its corresponding retractor muscles degenerate, indicating the transition from planktonic to benthic life. We observed a conserved pattern of larval musculature development and revealed a high degree of conservation across bivalve species, with comparable emergence times during myogenesis. Furthermore, exposure to the Hh signaling inhibitor cyclopamine impaired larval muscle development, reduced larval swimming activity, and inhibited larval metamorphosis in M. coruscus. Cyclopamine-mediated inhibition of Hh signaling led to reduced expression of four key genes within the Hh signaling pathway (McHh , McPtc , McSmo , and McGli) and the striated myosin heavy chain gene (McMHC). It is hypothesised that the abnormal larval muscle development in cyclopamine-treated groups may be an indirect effect due to disrupted McMHC expression. We provide evidence for the first time that cyclopamine treatment inhibited larval metamorphosis in bivalves, highlighting the potential involvement of Hh signaling in mediating larval muscle development and metamorphosis in M. coruscus. The present study provides insights into the dynamic nature of myogenesis and the regulatory role of the Hh signaling pathway during larval development and metamorphosis in M. coruscus. The results obtained in this study contribute to a better understanding of the evolutionary significance of Hh signaling in bivalves and shed light on the mechanisms underlying larval muscle development and metamorphosis in marine invertebrates. [Display omitted] • Studied larval myogenesis with specific muscle emergence and degeneration in Mytilus coruscus. • Identified conserved patterns of larval musculature across bivalve species. • Hh signaling inhibitor cyclopamine inhibited larval muscle development and larval metamorphosis. • Cyclopamine inhibited the expression of key genes within the Hh signaling and a myosin gene. [ABSTRACT FROM AUTHOR]

• Combined genetic-pharmacologic ADAMTS1 and ADAMTS5 inactivation in mice. • Degradomics identification of putative ADAMTS1 and ADAMTS5 substrates. • A requirement for versican proteolysis and glypican-6 in heart development. • Novel roles for ADAMTS1, ADAMTS5 and glypican-6 in hedgehog signaling in the heart. Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. We show that matrix-degrading metalloproteases ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each gene have mild cardiac anomalies, however, their combined genetic inactivation to elicit cooperative roles is precluded by tight gene linkage. Therefore, we coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and investigated their potential substrates in mouse cardiac development. ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows spanning myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant versican mutants. Mass spectrometry-based N-terminomics was used to seek relevant substrates. Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which was also observed in mice with ADAMTS cleavage-resistant versican. Combined inactivation after 12.5 days impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect. N-terminomics of combined ADAMTS knockout and control hearts identified a cleaved glypican-6 peptide only in the controls. ADAMTS1 and ADAMTS5 expression in cells was associated with specific glypican-6 cleavages. Paradoxically, combined ADAMTS1 and ADAMTS5 inactivation reduced cardiac glypican-6 and outflow tract Gpc6 transcription. Notably, Gpc6 −/− hearts demonstrated similar rotational defects as combined ADAMTS inactivated hearts and both had reduced hedgehog signaling. Thus, versican proteolysis in cardiac jelly at the canonical Glu441-Ala442 site is cooperatively mediated by ADAMTS1 and ADAMTS5 and required for proper ventricular cardiomyogenesis, whereas, reduced glypican-6 after combined ADAMTS inactivation impairs hedgehog signaling, leading to outflow tract malrotation. [ABSTRACT FROM AUTHOR]

Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent to the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the intrinsically disordered C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEFs) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia, and upregulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild-type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. The CILK1 A615T mutant protein was not increased to the same level as the wild-type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single-residue variant of CILK1 is compromised, and this impairs the maintenance of primary cilia and Hedgehog signaling. [ABSTRACT FROM AUTHOR]

A 62-year-old man presented with a slowly growing, painless lesion on his face. This eventually led to a progressive left-eye vision lesion, and the patient was subsequently diagnosed with advanced basal cell carcinoma (BCC). Of note, BCC involving cranial nerves is extremely rare, making this case unique and important to highlight. Standard treatment options for BCC involve surgery, radiation, or platinum-based chemotherapy. However, targeted therapies such as sonidegib and vismodegib - sonic hedgehog pathway inhibitors - have emerged that have been approved for treating BCC, as have anti-PD1 immunotherapies, such as cemiplimab, with their success likely based on the high tumor mutational burden seen in some of these tumors. Epidermal growth factor receptor (EGFR) inhibitors also serve a role in treating this condition as well. Molecular studies on metastatic/advanced BCC and other rare malignancies may inform treatment therapeutic decisions. [ABSTRACT FROM AUTHOR]

Cerebellar injury in preterm infants with central nervous system (CNS) hemorrhage results in lasting neurological deficits and an increased risk of autism. The impact of blood-induced pathways on cerebellar development remains largely unknown, so no specific treatments have been developed to counteract the harmful effects of blood after neurovascular damage in preterm infants. Here, we show that fibrinogen, a blood-clotting protein, plays a central role in impairing neonatal cerebellar development. Longitudinal MRI of preterm infants revealed that cerebellar bleeds were the most critical factor associated with poor cerebellar growth. Using inflammatory and hemorrhagic mouse models of neonatal cerebellar injury, we found that fibrinogen increased innate immune activation and impeded neurogenesis in the developing cerebellum. Fibrinogen inhibited sonic hedgehog (SHH) signaling, the main mitogenic pathway in cerebellar granule neuron progenitors (CGNPs), and was sufficient to disrupt cerebellar growth. Genetic fibrinogen depletion attenuated neuroinflammation, promoted CGNP proliferation, and preserved normal cerebellar development after neurovascular damage. Our findings suggest that fibrinogen alters the balance of SHH signaling in the neurovascular niche and may serve as a therapeutic target to mitigate developmental brain injury after CNS hemorrhage. [ABSTRACT FROM AUTHOR]

Lorlatinib is a pharmaceutical ALK kinase inhibitor used to treat ALK driven non-small cell lung cancers. This paper analyses the intersection of past published data on the physiological consequences of two unrelated drugs from general medical practice—itraconazole and cilostazol—with the pathophysiology of ALK positive non-small cell lung cancer. A conclusion from that data analysis is that adding itraconazole and cilostazol may make lorlatinib more effective. Itraconazole, although marketed worldwide as a generic antifungal drug, also inhibits Hedgehog signaling, Wnt signaling, hepatic CYP3A4, and the p-gp efflux pump. Cilostazol, marketed worldwide as a generic thrombosis preventative drug, acts by inhibiting phosphodiesterase 3, and, by so doing, lowers platelets' adhesion, thereby partially depriving malignant cells of the many tumor trophic growth factors supplied by platelets. Itraconazole may enhance lorlatinib effectiveness by (i) reducing or stopping a Hedgehog-ALK amplifying feedback loop, by (ii) increasing lorlatinib's brain levels by p-gp inhibition, and by (iii) inhibiting growth drive from Wnt signaling. Cilostazol, surprisingly, carries minimal bleeding risk, lower than that of aspirin. Risk/benefit assessment of the combination of metastatic ALK positive lung cancer being a low-survival disease with the predicted safety of itraconazole-cilostazol augmentation of lorlatinib favors a trial of this drug trio in ALK positive lung cancer. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases. [ABSTRACT FROM AUTHOR]

Background: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown. Methods: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling. Results: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force. Conclusions: Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer stem cells (BCSCs) are a small population of cells in breast cancer tumors, playing a putative role in the cancer's progression. BCSC biomarkers are associated with cells' enhanced growth, adhesion, migration, and invasion potential and are responsible for poor outcomes. Due to the vast heterogeneity of breast cancer signaling pathways and associated therapeutic targets, treatment strategies vary and depend on the molecular subtype of BC. Nevertheless, drugs affecting the Wnt, Notch, Hedgehog, PI3K/Akt/mTOR, and HER2 signaling pathways have been effectively applied in BCSC elimination strategies. The complexity of the characteristics of each molecular subtype of breast cancer, with an emphasis on the involvement of breast cancer stem cells in the development, metastasis, and recurrence of the tumor, requires greater understanding. Continuous updating of knowledge and the development of new therapies will allow the effective elimination of cancer cells and the rapid recovery of patients. Breast cancer is one of the most frequently detected malignancies worldwide. It is responsible for more than 15% of all death cases caused by cancer in women. Breast cancer is a heterogeneous disease representing various histological types, molecular characteristics, and clinical profiles. However, all breast cancers are organized in a hierarchy of heterogeneous cell populations, with a small proportion of cancer stem cells (breast cancer stem cells (BCSCs)) playing a putative role in cancer progression, and they are responsible for therapeutic failure. In different molecular subtypes of breast cancer, they present different characteristics, with specific marker profiles, prognoses, and treatments. Recent efforts have focused on tackling the Wnt, Notch, Hedgehog, PI3K/Akt/mTOR, and HER2 signaling pathways. Developing diagnostics and therapeutic strategies enables more efficient elimination of the tumor mass together with the stem cell population. Thus, the knowledge about appropriate therapeutic methods targeting both "normal" breast cancer cells and breast cancer stem cell subpopulations is crucial for success in cancer elimination. [ABSTRACT FROM AUTHOR]

Simple Summary: Honeybees (Apis mellifera) rely on their olfaction for various daily activities and tasks. Smoothened (Smo) serves as a critical element in the Hedgehog pathway and is likely associated with the modulation of the olfactory capabilities of bees. This study involved the identification, localization, and functional validation of Smo. The findings establish a foundational understanding of the role of Smo in influencing the olfactory abilities of bees. Smoothened (Smo) is a critical component regulating the Hedgehog signaling pathway. However, whether Smo is associated with the modulation of olfactory recognition capabilities of bees remains unclear. In this study, we amplified Smo from Apis mellifera. The coding sequence of Smo was 2952 bp long, encoded 983 amino acids. Smo was most highly expressed in the antennae. Cyclopamine (200 μg/mL) significantly reduced but purmorphamine (800 μg/mL) significantly increased Smo expression (p < 0.05). OR152 and OR2 expression in the cyclopamine group significantly decreased, whereas OR152 expression in the purmorphamine group significantly increased (p < 0.05). A significant decrease in the relative values of electroantennography was observed in the cyclopamine group exposed to neral. Behavioral tests indicated a significant decrease in the attractive rates of neral, VUAA1, linalool, and methyl heptenone in the cyclopamine group. Conversely, the selection rates of linalool and methyl heptenone in the purmorphamine group significantly increased. Our findings indicate that Smo may play a role in modulating olfactory receptors in bees. [ABSTRACT FROM AUTHOR]

Uterine leiomyosarcoma (uLMS) is the most common subtype of uterine sarcomas. They have a poor prognosis with high rates of recurrence and metastasis. The five-year survival for uLMS patients is between 25 and 76%, with survival rates approaching 10–15% for patients with metastatic disease at the initial diagnosis. Accumulating evidence suggests that several biological pathways are involved in uLMS pathogenesis. Notably, drugs that block abnormal functions of these pathways remarkably improve survival in uLMS patients. However, due to chemotherapy resistance, there remains a need for novel drugs that can target these pathways effectively. In this review article, we provide an overview of the recent progress in ascertaining the biological functions and regulatory mechanisms in uLMS from the perspective of aberrant biological pathways, including DNA repair, immune checkpoint blockade, protein kinase and intracellular signaling pathways, and the hedgehog pathway. We review the emerging role of epigenetics and epitranscriptome in the pathogenesis of uLMS. In addition, we discuss serum markers, artificial intelligence (AI) combined with machine learning, shear wave elastography, current management and medical treatment options, and ongoing clinical trials for patients with uLMS. Comprehensive, integrated, and deeper insights into the pathobiology and underlying molecular mechanisms of uLMS will help develop novel strategies to treat patients with this aggressive tumor. [ABSTRACT FROM AUTHOR]

During the morphological changes occurring in osteoblast differentiation, Sonic hedgehog (Shh) plays a crucial role. While some progress has been made in understanding this process, the epigenetic mechanisms governing the expression of Hh signaling members in response to bone morphogenetic protein 7 (BMP7) signaling in osteoblasts remain poorly understood. To delve deeper into this issue, we treated pre‐osteoblasts (pObs) with 100 ng/mL of BMP7 for up to 21 days. Initially, we validated the osteogenic phenotype by confirming elevated expression of well‐defined gene biomarkers, including Runx2, Osterix, Alkaline Phosphatase (Alp), and bone sialoprotein (Bsp). Simultaneously, Hh signaling‐related members Sonic (Shh), Indian (Ihh), and Desert (Dhh) Hedgehog (Hh) exhibited nuanced modulation over the 21 days in vitro period. Subsequently, we evaluated epigenetic markers, and our data revealed a notable change in the CpG methylation profile, considering the methylation/hydroxymethylation ratio. CpG methylation is a reversible process regulated by DNA methyltransferases and demethylases, including Ten‐eleven translocation (Tets), which also exhibited changes during the acquisition of the osteogenic phenotype. Specifically, we measured the methylation pattern of Shh‐related genes and demonstrated a positive Pearson correlation for GLI Family Zinc Finger 1 (Gli1) and Patched (Ptch1). This data underscores the significance of the epigenetic machinery in modulating the BMP7‐induced osteogenic phenotype by influencing the activity of Shh‐related genes. In conclusion, this study highlights the positive impact of epigenetic control on the expression of genes related to hedgehog signaling during the morphogenetic changes induced by BMP7 signaling in osteoblasts. [ABSTRACT FROM AUTHOR]

Objectives: Sonic hedgehog hepatocellular adenoma (shHCA) is a new hepatocellular adenoma (HCA) subgroup characterized by high risk of hemorrhage. ShHCA account for below 10% of all HCA cases and are often associated with female gender, obesity, and non-alcoholic steatohepatitis. No specific MRI characteristics have been described to date. The objective of this study was to assess the value of using MRI to identify shHCA, and correlate MRI findings with histology. Methods: We retrospectively collected MRI scans of 29 patients with shHCA from our center and from different liver referral centers to include 35 lesions. Diagnosis of shHCA was assessed by immunohistochemical overexpression of argininosuccinate synthase 1 or prostaglandin D2 synthase, then confirmed by molecular analysis of sonic hedgehog pathway activation and/or by proteomic analysis. Results: In 46% (n = 16/35) of shHCA cases, we detected intralesional fluid-filled cavities defined on MR images as fluid-like foci markedly hyperintense on T2-weighted sequences, and hypointense on T1-weighted sequences, with or without delayed enhancement. Pathologically, these cavities were observed in 54% of cases as vacuoles filled with blood at different stages of degradation. Hemorrhage and/or necrosis were detected among 71% of cases by MRI analysis (n = 25/35) versus 82% pathologically. Seventeen percent of shHCA cases (n = 6/35) were completely homogeneous via MRI and pathological analysis. No MRI criteria was found in favor of focal nodular hyperplasia, HNF1A-mutated HCA, or typical inflammatory HCA. Conclusion: We reveal the presence of intralesional fluid-filled cavities among 46% of our shHCA cases that represent a new MRI finding possibly helpful for shHCA diagnosis. Clinical relevance statement: This multicenter study is the first clinical study about the radiological aspect of this new hepatocellular adenoma subgroup. This highlights a strong correlation between MRI and histological analysis, with a specific pattern emerging for diagnosis. Key Points: • Sonic hedgehog hepatocellular adenoma is a new hepatocellular adenoma subgroup associated with high risk of hemorrhage, but imaging features of this subgroup remain unknown. • Analysis of MR images and correlation with pathology revealed intralesional fluid-filled cavities and necrotic-hemorrhagic changes. • Intralesional fluid-filled cavities have not yet been described in other adenoma subtypes and represent a new MRI finding for sonic hedgehog hepatocellular adenoma. [ABSTRACT FROM AUTHOR]

Introduction: Basal cell carcinoma (BCC) is treated with local surgery or noninvasive treatment modalities. If a BCC remains untreated, it can develop into a locally advanced BCC or a metastatic BCC. Case Presentation: Here we report in detail the management of three complex advanced BCC (aBCC) after treatment failure with vismodegib. On all tumors, next generation DNA sequencing in the Center for Personalized Cancer Treatment-02 (CPCT-02) study was performed; subsequently, patients were included in the Drug Rediscovery Protocol (DRUP) trial, in which treatment was started with commercially available targeted anticancer drugs based on the molecular tumor profile. All patients showed partial response or stable disease following treatment with second line PD-1 inhibitors with an average duration of response of 12.3 months. Discussion/Conclusion: Immunotherapy can be a treatment option for aBCC resistant to hedgehog pathway inhibitor treatment. However, despite the high tumor mutational burden of aBCCs, immunotherapy does not always lead to a long response. Rechallenge or combining treatment of hedgehog inhibitors and PD-1 inhibitors by parallel or alternating cycles may be a strategy to lengthen the treatment response. [ABSTRACT FROM AUTHOR]

The Hedgehog (HH) pathway is crucial for embryonic development, and adult homeostasis. Its dysregulation is implicated in multiple diseases. Existing cellular models used to study HH signal regulation in mammals do not fully recapitulate the complexity of the pathway. Here we show that Spinal Cord Organoids (SCOs) can be applied to quantitively study the activity of the HH pathway. During SCO formation, the specification of different categories of neural progenitors (NPC) depends on the intensity of the HH signal, mirroring the process that occurs during neural tube development. By assessing the number of NPCs within these distinct subgroups, we are able to categorize and quantify the activation level of the HH pathway. We validate this system by measuring the effects of mutating the HH receptor PTCH1 and the impact of HH agonists and antagonists on NPC specification. SCOs represent an accessible and reliable in-vitro tool to quantify HH signaling and investigate the contribution of genetic and chemical cues in the HH pathway regulation. [ABSTRACT FROM AUTHOR]

Histone demethylase JMJD2D (also known as KDM4D) can specifically demethylate H3K9me2/3 to activate its target gene expression. Our previous study has demonstrated that JMJD2D can protect intestine from dextran sulfate sodium (DSS)-induced colitis by activating Hedgehog signaling; however, its involvement in host defense against enteric attaching and effacing bacterial infection remains unclear. The present study was aimed to investigate the role of JMJD2D in host defense against enteric bacteria and its underlying mechanisms. The enteric pathogen Citrobacter rodentium (C. rodentium) model was used to mimic clinical colonic infection. The responses of wild-type and JMJD2D-/- mice to oral infection of C. rodentium were investigated. Bone marrow chimeric mice were infected with C. rodentium. JMJD2D expression was knocked down in CMT93 cells by using small hairpin RNAs, and Western blot and real-time PCR assays were performed in these cells. The relationship between JMJD2D and STAT3 was studied by co-immunoprecipitation and chromatin immunoprecipitation. JMJD2D was significantly up-regulated in colonic epithelial cells of mice in response to Citrobacter rodentium infection. JMJD2D-/- mice displayed an impaired clearance of C. rodentium, more body weight loss, and more severe colonic tissue pathology compared with wild-type mice. JMJD2D-/- mice exhibited an impaired expression of IL-17F in the colonic epithelial cells, which restricts C. rodentium infection by inducing the expression of antimicrobial peptides. Accordingly, JMJD2D-/- mice showed a decreased expression of β-defensin-1, β-defensin-3, and β-defensin-4 in the colonic epithelial cells. Mechanistically, JMJD2D activated STAT3 signaling by inducing STAT3 phosphorylation and cooperated with STAT3 to induce IL-17F expression by interacting with STAT3 and been recruited to the IL-17F promoter to demethylate H3K9me3. Our study demonstrates that JMJD2D contributes to host defense against enteric bacteria through up-regulating IL-17F to induce β-defensin expression. Author summary: Our study aimed to unravel the role of JMJD2D, a histone demethylase also known as KDM4D, in the host's defense against enteric bacterial infections and explore the underlying mechanisms. While we had previously demonstrated JMJD2D's protective effect in DSS-induced colitis by activating Hedgehog signaling, its involvement in defending against enteric attaching and effacing bacterial infections remained unexplored. To address this, we employed a Citrobacter rodentium (C. rodentium) infection model to mimic colonic infections in a clinical context. In response to C. rodentium infection, there was a notable increase in JMJD2D expression within the colonic epithelial cells of mice. Our investigation compared the responses of wild-type mice with those lacking JMJD2D (JMJD2D-/-) following oral C. rodentium infection. Notably, JMJD2D-/- mice exhibited impaired C. rodentium clearance, greater body weight loss, and more severe colonic tissue damage compared to their wild-type counterparts. Subsequent analysis revealed that JMJD2D deficiency resulted in reduced IL-17F expression in colonic epithelial cells. IL-17F is a known regulator that restricts C. rodentium infection by promoting the expression of antimicrobial peptides. Consequently, JMJD2D-/- mice showed diminished expression of critical antimicrobial peptides, including β-defensin-1, β-defensin-3, and β-defensin-4, in their colonic epithelial cells. Mechanistically, we discovered that JMJD2D played a pivotal role in activating STAT3 signaling by enhancing STAT3 phosphorylation. Moreover, JMJD2D collaborated with STAT3 to induce IL-17F expression. This cooperation involved JMJD2D physically interacting with STAT3 and binding to the IL-17F promoter, where it demethylated H3K9me3. In summary, our research reveals that JMJD2D contributes significantly to the host's defense against enteric bacterial infections by up-regulating IL-17F, which in turn induces the expression of antimicrobial peptides such as β-defensins. Our findings provide valuable insights into the molecular mechanisms governing host defense against enteric bacterial infections, emphasizing the potential therapeutic relevance of targeting JMJD2D in such contexts. [ABSTRACT FROM AUTHOR]

Objective: Unusual clinical course Background: A 52-year-old male patient presented with symptoms of chronic cough and persistent tracheal irritation 26 years after surgical closure of a tracheostoma, supported by an autologous auricular cartilage graft and cutaneous transplant. At the initial clinical presentation, the patient was an active smoker, with a cumulative dose of 31 pack years. Case Report: Bronchoscopy revealed endotracheal hair growth and local inflammation at the graft site. Initial anti-inflammatory, antimycotic, and antibacterial therapy was administered, followed by endoscopic structure remodeling. There were multiple recurrences with similar symptoms, showing isolated hair growth, without inflammation. Annual endoscopic restructuring sessions were indicated, and the patient experienced them as highly relieving. Recurrent hair growth was finally terminated by argon plasma laser-coagulation and after smoking cessation. We hypothesize that the onset of hair growth was triggered by the patient's cigarette smoking. Conclusions: Endotracheal hair growth is a potential complication of autograft-supported tracheal restructuring. The initial administration of antimicrobial and anti-inflammatory medication, combined with endoscopic restructuring, could have contained the active inflammation; the application of argon plasma laser-coagulation finally stopped the hair growth. Smoking is associated with the upregulation of molecular signaling pathways in the respiratory epithelium, which can stimulate hair follicles, such as sonic hedgehog protein, WNT-1/β-catenin, and epidermal growth factor receptor. [ABSTRACT FROM AUTHOR]

Simple Summary: Gorlin syndrome (GS) is a genetic disorder characterized by multiple basal cell carcinomas (BCCs) due to mutations in the hedgehog signaling pathway. Patients with GS may need dozens or even hundreds of surgical procedures in their lifetime, which can leave them severely scarred, deformed, and disfigured. In 16 patients with GS, we examined the effectiveness, safety, and length of response to oral hedgehog inhibitors. According to our retrospective study, sonidegib inhibited the growth of both newly diagnosed and pre-existing basal cell carcinomas more successfully and safely than vismodegib. Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome (GS), is a genetic disorder characterized by the development of multiple cutaneous BCCs due to mutations in the hedgehog signaling pathway. The use of hedgehog pathway inhibitors—vismodegib and sonidegib—has emerged as a promising therapeutic strategy for managing BCCs in individuals with GS. In a retrospective study conducted between March 2012 and January 2024, a cohort of 16 Gorlin syndrome patients who received treatment with either sonidegib or vismodegib were analyzed. The primary objectives of the study were to evaluate the efficacy, safety profile, and duration of response to oral hedgehog inhibitors in this patient population. The study assessed various parameters, including the number of new BCCs that developed before and after treatment initiation, the duration and sustainability of treatment responses, as well as the incidence of adverse effects associated with hedgehog inhibitor therapy. The findings of the study revealed that sustained treatment with hedgehog inhibitors could effectively suppress the progression of both new and existing BCCs. Furthermore, the results indicated that sonidegib exhibited superior efficacy and safety compared to vismodegib in the treatment of BCCs in individuals with GS. Notably, adjustments to the administration schedule of sonidegib were found to improve tolerability without compromising therapeutic efficacy, potentially leading to prolonged durations of treatment response and disease control. [ABSTRACT FROM AUTHOR]

Exposure to inorganic arsenic (As) is recognized as a risk factor for non-melanoma skin cancer (NMSC). We followed up with 7000 adults for 6 years who were exposed to As. During follow-up, 2.2% of the males and 1.3% of the females developed basal cell carcinoma (BCC), while 0.4% of the male and 0.2% of the female participants developed squamous cell carcinoma (SCC). Using a panel of more than 400 cancer-related genes, we detected somatic mutations (SMs) in the first 32 NMSC samples (BCC = 26 and SCC = 6) by comparing paired (tissue–blood) samples from the same individual and then comparing them to the SM in healthy skin tissue from 16 participants. We identified (a) a list of NMSC-associated SMs, (b) SMs present in both NMSC and healthy skin, and (c) SMs found only in healthy skin. We also demonstrate that the presence of non-synonymous SMs in the top mutated genes (like PTCH1, NOTCH1, SYNE1, PKHD1 in BCC and TP53 in SCC) significantly affects the magnitude of differential expressions of major genes and gene pathways (basal cell carcinoma pathways, NOTCH signaling, IL-17 signaling, p53 signaling, Wnt signaling pathway). These findings may help select groups of patients for targeted therapy, like hedgehog signaling inhibitors, IL17 inhibitors, etc., in the future. [ABSTRACT FROM AUTHOR]

The primary cilium, an antenna-like sensory organelle that protrudes from the surface of most eukaryotic cell types, has become a signaling hub of growing interest given that defects in its structure and/or function are associated with human diseases and syndromes, known as ciliopathies. With the continuously expanding role of primary cilia in health and diseases, identifying new players in ciliogenesis will lead to a better understanding of the function of this organelle. It has been shown that the primary cilium shares similarities with the immune synapse, a highly organized structure at the interface between an antigen-presenting or target cell and a lymphocyte. Studies have demonstrated a role for known cilia regulators in immune synapse formation. However, whether immune synapse regulators modulate ciliogenesis remains elusive. Here, we find that programmed death ligand 1 (PD-L1), an immune checkpoint protein and regulator of immune synapse formation, plays a role in the regulation of ciliogenesis. We found that PD-L1 is enriched at the centrosome/basal body and Golgi apparatus of ciliated cells and depleting PD-L1 enhanced ciliogenesis and increased the accumulation of ciliary membrane trafficking proteins Rab8a, BBS5, and sensory receptor protein PC-2. Moreover, PD-L1 formed a complex with BBS5 and PC-2. In addition, we found that depletion of PD-L1 resulted in the ciliary accumulation of Gli3 and the downregulation of Gli1. Our results suggest that PD-L1 is a new player in ciliogenesis, contributing to PC-2-mediated sensory signaling and the Hh signaling cascade. [ABSTRACT FROM AUTHOR]

The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development. [ABSTRACT FROM AUTHOR]