Does the sonic hedgehog signaling pathway play a role in anti-reflux mechanism of bladder in children?

OBJECTIVE: A hedgehog family ligand, namely, sonic hedgehog (SHH), was reported to be important in the development of bladder and ureter smooth muscle. In this prospective study, we aimed to determine protein expression of SHH in resected ureterovesical junction (UVJ) segments of children with vesicoureteral reflux (VUR). MATERIALS AND METHODS: The study group included 19 children; 12 (63%) girls, 7 (37%) boys, who had ureteroneocystostomy operation; 3 (15.7%) right sided, 7 (36.8%) left sided, 9 (47.3%) bilateral, due to primary VUR between years 2015 and 2018. Totally, 28 UVJ segments were examined for Western Blot analysis to determine related protein expression levels. RESULTS: The mean Western blot band area of SHH gene pathway related protein was 3880.69 (2059.55-13941.61) while the mean area of ß-Actin, the house-keeping gene, was 20180.25 (9530.39-26709.75) (p = 0.001). Correlation analyses between grade of reflux and protein expression of SHH gene pathways revealed no significant relation (p = 0.300). When the UV samples were grouped as low- and high-grade reflux and compared in terms of SHH protein expression levels, no statistically significant difference was found between groups (p = 0.818). CONCLUSION: We concluded that SHH signaling molecule which is effective in development of bladder and ureter smooth musculature might also be effective in etiopathology of reflux.

OBJETIVO: Se ha informado que el ligando sonic hedgehog (SHH) es importante en el desarrollo de los músculos lisos de la vejiga y el uréter. Nuestro objetivo fue determinar la expresión proteica de SHH en los segmentos de la unión ureterovesical de niños con reflujo vesicoureteral (RVU). MATERIALES Y MÉTODOS: El grupo de estudio incluyó a 19 niños; 12 (63%) niñas, 7 (37%) niños, que tuvieron operación de ureteroneocistostomía (UNC); 3 (15.7%) derecho, 7 (36.8%) izquierdo, 9 (47.3%) bilateral, por RVU primario entre los años 2015-2018. Se examinaron un total de 28 segmentos de la unión ureterovesical para análisis de transferencia Western para determinar los niveles de expresión de proteínas relacionadas en las muestras. RESULTADOS: El área media de la banda de transferencia Western de la proteína relacionada con la vía del gen SHH fue de 3880.69 (2059.55-13941.61), mientras que el área media de la ß-actina, el gen de limpieza, fue de 20180.25 (9530.39-26709.75) (p = 0.001). Los análisis de correlación entre el grado de reflujo y la expresión de proteínas de las vías del gen SHH no revelaron una relación significativa (p = 0.300). CONCLUSIÓN: Concluimos que la molécula de señalización SHH también podría ser efectiva en la etiopatología del reflujo vesicoureteral.

The First Patient with Tibial Hemimelia-Polysyndactyly-Triphalangeal Thumb Syndrome Caused by De Novo c.423+4916 T>C ZRS Variant: A Case Report.

Genetic variants in the zone of polarizing activity regulatory sequence (ZRS) that induce ectopic expression of the SHH gene have been associated with different ZRS-related phenotypes. We report the first patient with a de novo variant, c.423+4916 T>C, in ZRS (previously classified as a variant of uncertain significance) that causes tibial hemimelia-polysyndactyly-triphalangeal thumb syndrome (THPTTS). A two-month-old male patient presented with bilateral preaxial polydactyly, triphalangeal thumb, and tibial agenesis and was heterozygous for the variant c.423+4916T>C (neither of his parents was a carrier). The findings obtained from the family study were sufficient to reclassify the variant from "uncertain significance" to "likely pathogenic" according to three criteria from the American College of Medical Genetics and Genomics guidelines, as follows: (1) absence of gnomAD, (2) confirmation of paternity and maternity, and (3) strong phenotype-genotype association. In ZRS-associated syndromes, a wide clinical spectrum has been observed, ranging from polydactyly to THPTTS; our patient has the most severe and rare phenotype. We did not perform functional assays. However, the c.423+4916T>C variant is flanked by three variants, which have been proven not only to cause the phenotype but also to increase the expression of SHH. Through all this data gathering, we consider the c.423+4916T>C variant to be causative of THPTTS.

The genomes of all lungfish inform on genome expansion and tetrapod evolution.

The genomes of living lungfishes can inform on the molecular-developmental basis of the Devonian sarcopterygian fish-tetrapod transition. We de novo sequenced the genomes of the African (Protopterus annectens) and South American lungfishes (Lepidosiren paradoxa). The Lepidosiren genome (about 91 Gb, roughly 30 times the human genome) is the largest animal genome sequenced so far and more than twice the size of the Australian (Neoceratodus forsteri)1 and African2 lungfishes owing to enlarged intergenic regions and introns with high repeat content (about 90%). All lungfish genomes continue to expand as some transposable elements (TEs) are still active today. In particular, Lepidosiren's genome grew extremely fast during the past 100 million years (Myr), adding the equivalent of one human genome every 10 Myr. This massive genome expansion seems to be related to a reduction of PIWI-interacting RNAs and C2H2 zinc-finger and Krüppel-associated box (KRAB)-domain protein genes that suppress TE expansions. Although TE abundance facilitates chromosomal rearrangements, lungfish chromosomes still conservatively reflect the ur-tetrapod karyotype. Neoceratodus' limb-like fins still resemble those of their extinct relatives and remained phenotypically static for about 100 Myr. We show that the secondary loss of limb-like appendages in the Lepidosiren-Protopterus ancestor was probably due to loss of sonic hedgehog limb-specific enhancers.

Hedgehog components are overexpressed in a series of liver cancer cases.

Liver cancers, including hepatocellular carcinoma (HCC), are the sixth most common cancer and the third leading cause of cancer-related death worldwide, representing a global public health problem. This study evaluated nine patients with HCC. Six of the cases involved hepatic explants, and three involved hepatic segmentectomy for tumor resection. Eight out of nine tumors were HCC, with one being a combined hepatocellular-cholangiocarcinoma tumor. Conventional markers of hepatocellular differentiation (Hep Par-1, arginase, pCEA, and glutamine synthetase) were positive in all patients, while markers of hepatic precursor cells (CK19, CK7, EpCAM, and CD56) were negative in most patients, and when positive, they were detected in small, isolated foci. Based on in silico analysis of HCC tumors from The Cancer Genome Atlas database, we found that Hedgehog (HH) pathway components (GLI1, GLI2, GLI3 and GAS1) have high connectivity values (module membership > 0.7) and are strongly correlated with each other and with other genes in biologically relevant modules for HCC. We further validated this finding by analyzing the gene expression of HH components (PTCH1, GLI1, GLI2 and GLI3) in our samples through qPCR, as well as by immunohistochemical analysis. Additionally, we conducted a chemosensitivity analysis using primary HCC cultures treated with a panel of 18 drugs that affect the HH pathway and/or HCC. Most HCC samples were sensitive to sunitinib. Our results offer a comprehensive view of the molecular landscape of HCC, highlighting the significance of the HH pathway and providing insight into focused treatments for HCC.

Conditioned Medium From Reactive Astrocytes Inhibits Proliferation, Resistance, and Migration of p53-Mutant Glioblastoma Spheroid Through GLI-1 Downregulation.

Glioblastoma (GBM) aggressiveness is partly driven by the reactivation of signaling pathways such as Sonic hedgehog (SHH) and the interaction with its microenvironment. SHH pathway activation is one of the phenomena behind the glial transformation in response to tumor growth. The reactivation of the SHH signaling cascade during GBM-astrocyte interaction is highly relevant to understanding the mechanisms used by the tumor to modulate the adjacent stroma. The role of reactive astrocytes considering SHH signaling during GBM progression is investigated using a 3D in vitro model. T98G GBM spheroids displayed significant downregulation of SHH (61.4 ± 9.3%), GLI-1 (6.5 ± 3.7%), Ki-67 (33.7 ± 8.1%), and mutant MTp53 (21.3 ± 10.6%) compared to the CONTROL group when incubated with conditioned medium of reactive astrocytes (CM-AST). The SHH pathway inhibitor, GANT-61, significantly reduced previous markers (SHH = 43.0 ± 12.1%; GLI-1 = 9.5 ± 3.4%; Ki-67 = 31.9 ± 4.6%; MTp53 = 6.5 ± 7.5%) compared to the CONTROL, and a synergistic effect could be observed between GANT-61 and CM-AST. The volume (2.0 ± 0.2 × 107 µm³), cell viability (80.4 ± 3.2%), and migration (41 ± 10%) of GBM spheroids were significantly reduced in the presence of GANT-61 and CM-AST when compared to CM-AST after 72 h (volume = 2.3 ± 0.4 × 107 µm³; viability = 92.2 ± 6.5%; migration = 102.5 ± 14.6%). Results demonstrated that factors released by reactive astrocytes promoted a neuroprotective effect preventing GBM progression using a 3D in vitro model potentiated by SHH pathway inhibition.

New Smoothened ligands based on the purine scaffold as potential agents for treating pancreatic cancer.

Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 µM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 µM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.

PTCH1 gene variants rs357564, rs2236405, rs2297086 and rs41313327, mRNA and tissue expression in basal cell carcinoma patients from Western Mexico.

BACKGROUND: Basal cell carcinoma (BCC) represents about 80% of all cases of skin cancer. The PTCH1 is a transmembrane protein of the Sonic Hedgehog signaling pathway that regulates cell proliferation. Genetic variants in PTCH1 gene have been previously described in association with BCC development. In addition, PTCH1 mRNA and protein expression analysis are also significant to understand its role in skin cancer physiopathology. METHODS: An analytical cross-sectional study was performed, and a total of 250 BCC patients and 290 subjects from the control group (CG) were included, all born in western Mexico. The genotypes and relative expression of the mRNA were determined by TaqMan® assay. The protein expression was investigated in 70 BCC paraffin-embedded samples with PTCH1 antibodies. Semi-quantitative analysis was performed to determine the expression level in the immunostained cells. RESULTS: We did not find evidence of an association between PTCH1 rs357564, rs2297086, rs2236405, and rs41313327 genetic variants and susceptibility to BCC. Likewise, no statistically significant differences were found in the comparison of the mRNA level expression between BCC and CG (p > 0.05). The PTCH1 protein showed a low expression in 6 of the analyzed samples and moderate expression in 1 sample. No association was found between genetic variants, protein expression, and demographic-clinical characteristics (p > 0.05). CONCLUSION: The studied PTCH1 variants may not be associated with BCC development in the Western Mexico population. The PTCH1 mRNA levels were lower in patients with BCC compared to the control group, but its protein was underexpressed in the tissue samples.

Development of the Goose Tongue Filiform Papillae: Could it be Tooth-Like Sense Organs? / Desarrollo de las Papilas Filiformes de la Lengua de Ganso: ¿Podrían ser Órganos Sensoriales Parecidos a los Dientes?

SUMMARY: The geese's tongue filiform papillae are particularly long, and exhibit the same morphology of a tooth, evoking the lingual teeth of several fishes. In adult animals, they contain numerous mechanical Herbst's corpuscles but no taste buds. In the embryo, they appear since stage 38 and acquire their definitive shape between stages 38 and 42. They express several proteins associated with mammalian tooth development (BMP4, β-catenin, SHH, PITX2, PAX9), also known to be linked to parrot's pseudoteeth and goose's denticulations development. Neurofilaments are early present in the papillae primordia, and appear particularly numerous in adult papillae. Our results suggest that these papillae constitute a mechanical organ with a « tooth shape » derived from ancestral odontodes, whose development is controlled by numerous genes involved in classical odontogenesis.

Las papilas filiformes de la lengua de los gansos son particularmente largas y exhiben la morfología de un diente, evocando los dientes linguales presentes en varios peces. En los animales adultos, contienen numerosos corpúsculos de Herbst mecánicos, aunque una ausencia de papilas gustativas. En el embrión, aparecen a partir del estadio 38 y adquieren su forma definitiva entre los estadios 38 y 42. Expresan varias proteínas asociadas al desarrollo dentario de los mamíferos (BMP4, β-catenina, SHH, PITX2, PAX9), también conocidas por estar asociadas al desarrollo de pseudodientes en el loro y denticulaciones en el ganso. Los neurofilamentos están presentes tempranamente en los primordios de las papilas y aparecen particularmente numerosos en las papilas adultas. Nuestros resultados sugieren que estas papilas constituyen un órgano mecánico con «forma de diente» derivado de odontoides ancestrales, cuyo desarrollo está controlado por numerosos genes implicados en la odontogénesis clásica.

Prechordal structures act cooperatively in early trabeculae development of gnathostome skull.

The vertebrate skull is formed by mesoderm and neural crest (NC) cells. The mesoderm contributes to the skull chordal domain, with the notochord playing an essential role in this process. The NC contributes to the skull prechordal domain, prompting investigation into the embryonic structures involved in prechordal neurocranium cartilage formation. The trabeculae cartilage, a structure of the prechordal neurocranium, arises at the convergence of prechordal plate (PCP), ventral midline (VM) cells of the diencephalon, and dorsal oral ectoderm. This study examines the molecular participation of these embryonic structures in gnathostome trabeculae development. PCP-secreted SHH induces its expression in VM cells of the diencephalon, initiating a positive feedback loop involving SIX3 and GLI1. SHH secreted by the VM cells of the diencephalon acts on the dorsal oral ectoderm, stimulating condensation of NC cells to form trabeculae. SHH from the prechordal region affects the expression of SOX9 in NC cells. BMP7 and SHH secreted by PCP induce NKX2.1 expression in VM cells of the diencephalon, but this does not impact trabeculae formation. Molecular cooperation between PCP, VM cells of the diencephalon, and dorsal oral ectoderm is crucial for craniofacial development by NC cells in the prechordal domain.

Low Expression of the NRP1 Gene Is Associated with Shorter Overall Survival in Patients with Sonic Hedgehog and Group 3 Medulloblastoma.

Medulloblastoma (MB) is the most common type of malignant pediatric brain tumor. Neuropilin-1 (NRP1), encoded by the NRP1 gene, is a transmembrane glycoprotein overexpressed in several types of cancer. Previous studies indicate that NRP1 inhibition displays antitumor effects in MB models and higher NRP1 levels are associated with poorer prognosis in MB patients. Here, we used a large MB tumor dataset to examine NRP1 gene expression in different molecular subgroups and subtypes of MB. We found overall widespread NRP1 expression across MB samples. Tumors in the sonic hedgehog (SHH) subgroup showed significantly higher NRP1 transcript levels in comparison with Group 3 and Group 4 tumors, with SHH samples belonging to the α, ß, Δ, and γ subtypes. When all MB subgroups were combined, lower NRP1 expression was associated with significantly shorter patient overall survival (OS). Further analysis showed that low NRP1 was related to poorer OS, specifically in MB subgroups SHH and Group 3 MB. Our findings indicate that patients with SHH and Group 3 tumors that show lower expression of NRP1 in MB have a worse prognosis, which highlights the need for subgroup-specific investigation of the NRP1 role in MB.

Taurine promotes axonal sprouting via Shh-mediated mitochondrial improvement in stroke.

PURPOSE: Motor function is restored by axonal sprouting in ischemic stroke. Mitochondria play a crucial role in axonal sprouting. Taurine (TAU) is known to protect the brain against experimental stroke, but its role in axonal sprouting and the underlying mechanism are unclear. METHODS: We evaluated the motor function of stroke mice using the rotarod test on days 7, 14, and 28. Immunocytochemistry with biotinylated dextran amine was used to detect axonal sprouting. We observed neurite outgrowth and cell apoptosis in cortical neurons under oxygen and glucose deprivation (OGD), respectively. Furthermore, we evaluated the mitochondrial function, adenosine triphosphate (ATP), mitochondrial DNA (mtDNA), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PCG-1α), transcription factor A of mitochondria (TFAM), protein patched homolog 1 (PTCH1), and cellular myelocytomatosis oncogene (c-Myc). RESULTS: TAU recovered the motor function and promoted axonal sprouting in ischemic mice. TAU restored the neuritogenesis ability of cortical neurons and reduced OGD-induced cell apoptosis. TAU also reduced reactive oxygen species, stabilized mitochondrial membrane potential, enhanced ATP and mtDNA content, increased the levels of PGC-1α, and TFAM, and restored the impaired levels of PTCH1, and c-Myc. Furthermore, these TAU-related effects could be blocked using an Shh inhibitor (cyclopamine). CONCLUSION: Taurine promoted axonal sprouting via Shh-mediated mitochondrial improvement in ischemic stroke.

Impact of neuroinflammation on epigenetic transcriptional control of Sonic Hedgehog members in the central nervous system.

Sonic Hedgehog (Shh) signaling plays a critical role during central nervous system (CNS) development, and its dysregulation leads to neurological disorders. Nevertheless, little is known about Shh signaling regulation in the adult brain. Here, we investigated the contribution of DNA methylation on the transcriptional control of Shh signaling pathway members and its basal distribution impact on the brain, as well as its modulation by inflammation. The methylation status of the promoter regions of these members and the transcriptional profile of DNA-modifying enzymes (DNA Methyltransferases - DNMTs and Tet Methylcytosine Dioxygenase - TETs) were investigated in a murine model of neuroinflammation by qPCR. We showed that, in the adult brain, methylation in the CpG promoter regions of the Shh signaling pathway members was critical to determine the endogenous differential transcriptional pattern observed between distinct brain regions. We also found that neuroinflammation differentially modulates gene expression of DNA-modifying enzymes. This study reveals the basal transcriptional profile of DNMTs and TETs enzymes in the CNS and demonstrates the effect of neuroinflammation on the transcriptional control of members of the Shh Signaling pathway in the adult brain.

Activation of Hedgehog signaling by the oncogenic RELA fusion reveals a primary cilia-dependent vulnerability in supratentorial ependymoma.

BACKGROUND: Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. METHODS: Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. RESULTS: Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. CONCLUSION: Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

Reversibility of digit loss revisited: Limb diversification in Bachia lizards (gymnophthalmidae).

Strict interpretations of the Dollo's Law lead to postulation that trait loss is irreversible and organisms never recover ancestral phenotypes. Dollo, however, admitted the possibility of reversals in trait loss when predicted differences between reversed (derived) and ancestral forms. Phenotypic signatures from reversals are expected, as the historical context of a reversal in trait loss differs from the initial setting where the trait originally evolved. This article combines morphological and molecular information for Bachia scolecoides to discuss phenotypic and genetic patterns established during processes that reversed digit loss in Gymnophthalmidae (also termed microteiid lizards). Results suggest that pathways leading to the derived tetradactyl state of B. scolecoides comprise particularities in their origin and associated processes. Autopodial bones of B. scolecoides lack digit identity, and muscle anatomy is very similar between manus and pes. Gymnophthalmidae sequence patterns in the limb-specific sonic hedgehog enhancer (ZRS) suggest that regulation of shh expression did not degenerate in Bachia, given the prediction of similar motifs despite mutations specific to Bachia. Persistence of developmental mechanisms might explain intermittent character expression leading to reversals of digit loss, as ZRS signaling pathways remain active during the development of at least one pair of appendices in Bachia, especially if some precursors persisted at early stages. Patterns of ZRS sequences suggest that irreversibility of trait loss might be lineage-specific (restricted to Gymnophthalmini) and contingent to the type of signature established. These results provide insights regarding possible mechanisms that may allow reactivation of developmental programs in specific regions of the embryo.

Sex differences in maternal odor preferences and brain levels of GAP-43 and sonic hedgehog proteins in infant SHR and Wistar Kyoto rats.

Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder that presents sex differences in the severity and presentation of symptoms, whose neurobiological basis is still unknown. Both Growth-associated Protein 43 (GAP-43) and Sonic hedgehog (Shh) are considered essential proteins for the appropriate brain development, but their participation in ADHD neurobiology have not been investigated yet. In this study, we hypothesized that alterations in these proteins could be related to behavioral traits to ADHD phenotype. Thus, both sexes of infant Spontaneously hypertensive rats (SHR, used as ADHD animal model) were evaluated for developmental milestones, locomotor activity, olfactory and recognition memory. Both GAP-43 and Shh were assessed in the olfactory bulb, frontal cortex and hippocampus in early and late infancy. During early infancy, SHR reached three developmental milestones later, and females showed olfactory memory impairment accompanied by increased levels of Shh in the olfactory bulb. In later infancy, hyperlocomotion, impaired recognition memory, and decreased Shh in the hippocampus were observed in SHR from both sexes. While in early infancy GAP-43 was not altered, it was decreased in the frontal cortex and hippocampus of female SHR in late infancy. Therefore, both Shh and GAP-43 are involved in the sex-dependent behavioral alterations showed by infant SHR. Despite the disorder's complexity and heterogeneity, our findings reveal important developmental parameters during SHR development and also emphasizes the relevance of studying sex differences in the ADHD context.

Immunohistochemical analysis of SHH, SMO and GLI-1 proteins in epithelial odontogenic lesions.

The present study analyzed the expression of proteins involved in the sonic hedgehog signaling pathway (SHH, SMO, and GLI-1) in benign epithelial odontogenic lesions (odontogenic keratocyst - OKC, ameloblastoma - AB, and adenomatoid odontogenic tumor - AOT) in order to identify the role of these proteins in the pathogenesis of these lesions. The sample consisted of 20 OKCs, 20 ABs, and 10 AOTs. The Kruskal-Wallis, Mann-Whitney U, and Spearman's (r) tests were used for statistical analysis, with the level of significance set at 5% (p < 0.05). The membrane/cytoplasmic expression of SHH was significantly higher in AB compared to AOT (p = 0.022) and OKC (p = 0.02). No differences were found in the membrane/cytoplasmic expression of SMO between the lesions studied. Regarding GLI-1, significant differences were observed at the nuclear level for AB and OKC compared to AOT (p < 0.0001). In addition, significant positive correlations were found between cytoplasmic and nuclear GLI-1 in AB (r = 0.482; p = 0.031) and OKC (r = 0.865; p < 0.0001), and between membrane/cytoplasmic SMO and cytoplasmic GLI-1 in AOT (r = 0.667; p = 0.035) and OKC (r = 0.535; p = 0.015). The results of this study confirm the participation of the sonic hedgehog signaling pathway in the pathogenesis of the lesions studied. Overexpression of SHH in ABs and nuclear expression of GLI-1 in ABs and OKCs indicate that these proteins contribute to the more aggressive behavior of these two lesions when compared to AOT.

Effects of long-term exposure to MST-312 on lung cancer cells tumorigenesis: Role of SHH/GLI-1 axis.

Replicative immortality is a key feature of cancer cells and it is maintained by the expression of telomerase, a promising target of novel therapies. Long-term telomerase inhibition can induce resistance, but the mechanisms underlying this process remain unclear. The Sonic hedgehog pathway (SHH) is an embryogenic pathway involved in tumorigenesis and modulates the transcription of telomerase. We evaluated the effects of long-term treatment of the telomerase inhibitor MST-312 in morphology, proliferation, resistance, and in the SHH pathway molecules expression levels in lung cancer cells. Cells treated for 12 weeks with MST-312 showed changes in morphology, such as spindle-shaped cells, and a shift in the distribution of F-ACTIN from cortical to diffuse. Treatment also significantly reduced cells' efficiency to form spheroids and their clonogenic potential, independently of the cell cycle and telomeric DNA content. Moreover, GLI-1 expression levels were significantly reduced after 12 weeks of MST-312 treatment, indicating a possible inhibition of this signaling axis in the SHH pathway, without hindering NANOG and OCT4 expression. Here, we described a novel implication of long-term treatment with MST-312 functionally and molecularly, shedding new light on the molecular mechanisms of this drug in vitro.

G9a/EHMT2 is a Potential Prognostic Biomarker and Molecular Target in SHH Medulloblastoma.

Changes in epigenetic programming are associated with cancer development during childhood. Components of the epigenetic machinery involved in normal embryonic development and hijacked by pediatric cancers include enzymes mediating post-translational modifications of DNA and histones that regulate chromatin structure, such as histone methyltransferases (HMTs). Overexpression of the HMT G9a (euchromatic histone lysine methyltransferase 2, EHMT2) has been described in several cancer types. Medulloblastoma (MB), the main type of malignant brain tumor afflicting children, is currently classified into four molecular subgroups. Here, we show that expression level of the G9a/Ehmt2 gene is higher in MB tumors belonging to the SHH, Group 3, and Group 4 subgroups, compared to Wnt tumors. Remarkably, high G9a expression was significantly associated with shorter overall survival in MB patients. We also present evidence that G9a inhibition dose-dependently reduces MB cell viability. Our findings suggest that higher transcription of G9a may be a predictor of poor prognosis in patients with SHH MB, and that inhibiting G9a activity can display antitumor effects in MB.

O papel de proteínas das vias WNT/ß-Catenina, TGF-ß/BMP4 e SHH na odontogênese e odontomas / The role of proteins from the WNT/ß-Catenin, TGF-ß/BMP4 and SHH pathways in odontogenesis and odontomas

O desenvolvimento do dente depende de uma série de interações sinalizadoras recíprocas entre o epitélio oral (EO) e o ectomesênquima derivado da crista neural, a via WNT com o TGF-ß e BMP4 tem sido implicada na tumorigênese. A via de sinalização tipo Wingless (Wnt) / ß-catenina é essencial para a ativação precoce da odontogênese e no desenvolvimento de tumores odontogênicos. O TGF-ß e as BMPs tem sido associadas aos processos de dentinogênese reacionária e reparadora. A sinalização de Shh pode regular a proliferação celular no ectomesênquima dentário, controlando assim a morfogênese dentária. O objetivo da pesquisa foi investigar a atuação de algumas proteínas das vias na odontogênese e na formação de odontomas e tumores odontogênicos mistos benignos, para isto, foi desenvolvido um estudo seccional restrospectivo e imuno-histoquímico contendo 23 odontomas compostos, 21 odontomas complexos, 17 germes dentários, 05 fibro-odontomas ameloblásticos e 01 fibroma ameloblástico. Os resultados encontrados demonstraram maiores imunoexpressões da via WNT/ß-catenina no epitélio dos germes dentários (p<0,001) e no fibroma ameloblástico, enquanto que, esteve no ectomesênquima dos odontomas (p<0,001) e fibro-odontomas ameloblásticos. A via WNT/ßcatenina correlacionou-se moderadamente e significativamente com a CK14 no epitélio (p = 0,007) dos odontomas. A BMP4 foi imunoexpressa, especialmente, no ectomesênquima dos odontomas complexos (mediana = 33,7; p<0,001). A via Shh foi mais imunoexpressa no epitélio dos germes dentários (p<0,001) e no ectomesênquima dos odontomas complexos (p=0,029). De forma similar, o TGFß apresentou maior imunoexpressão no epitélio dos germes dentários (p<0,001) e no ectomesênquima dos odontomas complexos (p = 0,002). O dente em desenvolvimento exibiu maiores concentrações para estas proteínas no epitélio odontogênico nas fases de botão e capuz e a expressão diferencial ocorreu, principalmente, no ectomesênquima dos tumores, o que indica que esse componente é de fato mais proliferativo (AU).

Tooth development depends on a series of reciprocal signaling interactions between oral epithelium (EO) and neural crest-derived ectomesenchyme, the WNT pathway with TGF-ß and BMP4 has been implicated in tumorigenesis. The Wingless (Wnt)/ß-catenin signaling pathway is essential for the early activation of odontogenesis and the development of odontogenic tumors. TGF-ß and BMPs have been associated with reactionary and reparative dentinogenesis processes. Shh signaling can regulate cell proliferation in dental ectomesenchyme, thus controlling dental morphogenesis. The objective of the research was to investigate the role of some proteins in the pathways in odontogenesis and in the formation of odontomas and benign mixed odontogenic tumors. tooth germs, 05 ameloblastic fibro-odontomas and 01 ameloblastic fibroma. The results found showed higher immunoexpressions of the WNT/ß-catenin pathway in the epithelium of tooth germs (p<0.001) and in ameloblastic fibroma, while it was in the ectomesenchyme of odontomas (p<0.001) and ameloblastic fibroodontomas. The WNT/ß-catenin pathway correlated moderately and significantly with CK14 in the epithelium (p = 0.007) of odontomas. BMP4 was immunoexpressed, especially in the ectomesenchyme of complex odontomas (median = 33.7; p<0.001). The Shh pathway was more immunoexpressed in the epithelium of tooth germs (p<0.001) and in the ectomesenchyme of complex odontomas (p=0.029). Similarly, TGF-ß showed higher immunoexpression in the epithelium of tooth germs (p<0.001) and in the ectomesenchyme of complex odontomas (p = 0.002). The developing tooth exhibited higher concentrations of these proteins in the odontogenic epithelium in the bud and cap phases and the differential expression occurred mainly in the ectomesenchyme of the tumors, which indicates that this component is in fact more proliferative (AU).