Smoothened Gene Research Articles

Expression of signaling molecules in ameloblastoma using cDNA microarray: A systematic review

Aim: Ameloblastoma is a benign but locally aggressive epithelial odontogenic tumor. The present review aimed to identify the most commonly expressed signaling molecule in ameloblastoma detected using cDNA microarray. Materials and Methods: It is a systematic review. In this review, articles were searched using databases—PubMed and Google Scholar and were also searched using Hand Search. All the studies which used cDNA microarray to detect the expression of signaling molecules were included. All the variants of ameloblastoma were included for the review. A total of 22 ameloblastoma cases were included in this systematic review. Out of three studies, 67% of the cases demonstrated upregulation of smoothened (SMO) and sonic hedgehog (SHH). Results: Out of three studies, 33% of the cases showed upregulation of toll-like receptor 2 (TLR2), fos proto-oncogene (FOS), Wnt family member 10a (Wnt10a), and patched and down-regulation of insulin-like growth factor 2 (IGF2). Of the 67% of the studies which described SMO and SHH, significant expression of SMO (73 times fold change) was noted. SHH expression was noted to have 0.88 times fold change. Of the 33% of the studies included, the highest average fold change was noted to be associated with FOS (14-fold change time) followed by Wnt10a (5.1-fold change times) and TLR2 (3.29-fold change times). However, downregulation of IGF2 by 0.09-fold change times was observed in 33% of the studies. Conclusion: The SMO gene from the SHH pathway was the highest expressed signaling molecule in ameloblastoma.

Chemically modified small interfering RNA targeting Hedgehog signaling pathway for rheumatoid arthritis therapy

Rheumatoid arthritis (RA) is an inflammatory disease that leads to disability; however, existing therapies are still unsatisfactory. Activated fibroblast-like synoviocytes (FLSs) play an essential role in synovitis formation and joint destruction in RA. The Hedgehog signaling pathway is aberrantly activated and contributes to the aggressive phenotype of RA-FLSs. However, it remains uncertain whether inhibiting Smoothened (SMO), a critical component of the Hedgehog signaling pathway, is an effective treatment for RA. Here, we design a series of small interfering RNAs (siRNAs) that specifically target the SMO gene. With precise chemical modifications, siRNAs' efficacy and stability are significantly improved, and the off-target effects are minimized. The optimized chemically modified siRNA (si-S1A3-Chol) decreases RA-FLS proliferation and invasiveness without the transfection reagent. Furthermore, si-S1A3-Chol injected intra-articularly effectively alleviates joint destruction and improves motor function in collagen-induced arthritis mouse models. Consequently, our results demonstrate that chemically modified siRNA targeting the Hedgehog signaling pathway may be a potential therapy for RA.

Ursolic acid inhibits the activation of smoothened-independent non-canonical hedgehog pathway in colorectal cancer by suppressing AKT signaling cascade.

It is being brought to light that smoothened (SMO)-independent non-canonical Hedgehog signaling is associated with the pathogenesis of various cancers. Ursolic acid (UA), a pentacyclic triterpenoid present in many medicinal herbs, manifests potent effectiveness against multiple malignancies including colorectal cancer (CRC). In our previous study, UA was found to protect against CRC in vitro by suppression of canonical Hedgehog signaling cascade. Here, the influence of UA on SMO-independent non-canonical Hedgehog signaling in CRC was investigated in the present study, which demonstrated that UA hampered the proliferation and migration, induced the apoptosis of HCT-116hSMO- cells with SMO gene knockdown, accompanied by the augmented expression of the suppressor of fused (SUFU), and lessened levels of MYC (c-Myc), glioma-associated oncogene (GLI1) and Sonic Hedgehog (SHH), and lowered phosphorylation of protein kinase B (PKB, AKT), suggesting that UA diminished non-canonical Hedgehog signal transduction in CRC. In HCT-116hSMO- xenograft tumor, UA ameliorated the symptoms, impeded the growth and caused the apoptosis of CRC, with heightened SUFU expression, and abated levels of MYC, GLI1, and SHH, and mitigated phosphorylation of AKT, indicating that UA down-regulated non-canonical Hedgehog signaling cascade in CRC. Taken together, UA may alleviate CRC by suppressing AKT signaling-dependent activation of SMO-independent non-canonical Hedgehog pathway.

Bovine SMO gene polymorphism in Anatolian Black Cattle and Anatolian Water Buffaloes

The purpose of this study was to determine the G>C mutation on exon 9 (G21234C) of Bovine Smoothened (SMO) gene polymorphism by using Cfr13I restriction enzyme with PCR-RFLP method in both Anatolian Black cattle and Anatolian Water Buffaloes. According to the G>C mutation on exon 9 (G21234C) of SMO gene, the results showed that there were three genotypes such as GG, GC and CC in Anatolian Black cattle, but there was only one genotype GG in Anatolian Water Buffaloes. Allele and genotype frequencies in Anatolian Black cattle were estimated as 0.35 and 0.65 for G and C alleles and 0.22, 026 and 0.52 for GG, GC and CC genotypes, respectively. The Chi-square test showed that the Anatolian Black cattle population was not in Hardy-Weinberg equilibrium with respect to G21234C substitution (PC mutation on exon 9 (G21234C) of SMO gene is polymorphic in cattle, it can be concluded that SMO gene is a potential candidate gene. However, such a statement cannot be expressed for buffaloes since it is monomorphic in Anatolian buffaloes.

Genetic and Epigenetic Regulation of the Smoothened Gene (SMO) in Cancer Cells.

(1) Background: The hedgehog (HH) signaling pathway is a key regulator of embryonic patterning, tissue regeneration, stem cell renewal, and cancer growth. The smoothened (SMO) protein regulates the HH signaling pathway and has demonstrated oncogenic activity. (2) Methods: To clarify the role of the HH signaling pathway in tumorigenesis, the expression profile of key HH signaling molecules, including SMO, PTCH1, GLI1, GLI2, and GLI3, were determined in 33 cancer cell lines and normal prostate cells and tissues. We performed a computational analysis of the upstream region of the SMO gene to identify the regulatory elements. (3) Results: Three potential CpG islands and several putative SMO promoter elements were identified. Luciferase reporter assays mapped key SMO promoter elements, and functional binding sites for SP1, AP1, CREB, and AP-2α transcription factors in the core SMO promoter region were confirmed. A hypermethylated SMO promoter was identified in several cancer cell lines suggesting an important role for epigenetic silencing of SMO expression in certain cancer cells. (4) Discussion: These results have important implications for our understanding of regulatory mechanisms controlling HH pathway activity and the molecular basis of SMO gene function. Moreover, this study may prove valuable for future research aimed at producing therapeutic downregulation of SMO expression in cancer cells.

Vismodegib Treatment in Patients with Advanced Basal Cell Carcinoma: LongTerm Efficacy and Safety Data with Focus on Secondary Resistance and Underlying Resistance Mechanism

Background: The smoothened inhibitor vismodegib is an effective targeted therapy for basal cell carcinoma (BCC) with a manageable and consistent safety profile. The occurrence of secondary resistance during treatment is a major problem and is associated with hedgehog pathway reactivation, predominantly through Smoothened (SMO) gene mutations. Objectives: To analyse efficacy and safety data after long-term follow-up of patients treated with vismodegib for advanced BCC in the University Hospital of Leuven, focusing on underlying genetic mechanisms of primary and secondary resistance to vismodegib. Methods: Twenty seven patients were retrospectively included in the study. We performed targeted sequencing of hedgehog pathway genes in 7 tumor samples from 4 patients with primary or secondary resistance to vismodegib. Results: Mean duration of follow-up was 29.9 months (1-77.7 months). Mean treatment duration was 13.3 months (1-64.5 months). The response rate to vismodegib was 93% (25/27 patients), with a partial response in 18/27 patients and a complete response in 7/27 patients. One patient maintained a complete response up to >3 years after vismodegib discontinuation. Six out of 27 patients (24%) developed secondary resistance during treatment, in three of them we detected acquired pathogenic SMO mutations in resistant tumor tissue. One patient with Bazex–Dupré–Christol syndrome showed primary resistance to vismodegib. No unexpected safety signals were detected in our analysis, however progression of Multiple Sclerosis (MS) was observed in one patient. Conclusions: We deliver new data about the response duration after vismodegib discontinuation and describe MS progression as a possible new adverse event to vismodegib. We describe for the first time vismodegib treatment and primary resistance to vismodegib in a patient with Bazex–Dupré–Christol syndrome. We highlight the problem of occurrence of secondary resistance in >20% of responders and confirm the previously reported resistance mechanism through acquired SMO mutations.

An Investigation of Polymorphism in <i>SMO</i> and <i>LMF</i>1 Genes and Their Association with Body Size in White Fulani and Muturu Cattle Breeds

Cattle production is integral to the people of Africa and her economy. To improve cattle productivity, there is a need to inculcate molecular marker-assisted selection into current breeding practices. In this study, we investigated, in White Fulani and Muturu cattle breeds, polymorphism of two candidate genes (Smoothened—SMO and Lipase Maturation Factor 1—LMF1) that have been reported to influence body size in cattle. The SMO gene encodes for the SMO (smoothened) receptor protein; a membrane signal transducer that plays a role in the control of bone formation and adipogenesis. LMF1 encodes for the LMF1 protein which regulates post-translational maturation of vascular lipases. Desired regions of the genes were amplified and genotyped by direct DNA sequencing or Polymerase chain reaction—Restriction Fragment Length Polymorphism. For association study, linear body measurements traits (BMTs) that can be used to predict body size were measured on each animal and values of BMTs measured were compared between both breeds. Very significant (P SMO while LMF1 was monomorphic at the expected (T > C) exon 4 mutation site. Association analysis revealed that polymorphism on G21234C, C22424T, T22939C and T23458G loci significantly affected BMTs (P SMO gene could be a useful candidate gene to facilitate marker assisted selection for body size while LMF1 is monomorphic in the cattle breeds.

Association between SNPs and Loss of Methylation Site on the CpG island of the Promoter Region of the Smoothened Gene, Potential Molecular Markers for Susceptibility to the Development of Basal Cell Carcinoma in the Brazilian Population.

Objective:Perform genotyping of SNPs in the promoter region of the SMO gene in BCC samples from patients from northeastern Brazil, and to determine if there is an association of these SNPs of the gene in question with the susceptibility to the development of the BCC. Methods:100 samples of paraffined tissue from patients with histopathological diagnosis of BCC and 100 control samples were analyzed for each polymorphism by a newly developed genotyping method, the Dideoxy Single Allele Specific – PCR. The software Bioestat - version 5.3 and Haploview 4.2 were used for the statistical analysis. For all tests a P-value <0.05 was considered significant. Results:The SNP rs538312246 is the Hardy-Weinberg equilibrium, therefore, it did not present significant association with the BCC (X² =2.343 and P<0.158). However, the CpG-SNPs rs375350898 and rs75827493 were significantly associated to the BCC in the analyzed samples (X2 = 27,740/21,500 and P <0001), the SNP rs75827493 showed a significant association with the BCC of the nodular subtype (P <0.0069). Therefore, our results suggest that SNPs rs375350898 and rs75827493 are potential molecular markers for susceptibility to BCC. Conclusion:The ability to detect SNP in a population, especially in promoter regions, has profoundly changed human genetic studies. This study allowed the understanding of the relationship between the presence of SNPs in CpG islands of the promoter region of the SMO gene can modify the methylation pattern and provide susceptibility to BCC in the population.

Attenuation of Post-Traumatic Osteoarthritis After Anterior Cruciate Ligament Injury Via Inhibition of Hedgehog Signaling.

We aimed to investigate whether post-traumatic osteoarthritis (PTOA) progression is appropriately represented by a PTOA mouse model using a unique climbing cage to add mechanical loading after anterior cruciate ligament (ACL) transection and to determine how Hedgehog signaling inhibition prevents PTOA progression by observing time-dependent morphological changes. This controlled laboratory study histologically compared mice with surgically-induced ACL transection (ACLT) and those with voluntary increased activity in a climbing cage from 1 week postoperatively (ACLT + climbing). We generated conditional knockout (cKO) mice with a deleted Smoothened (Smo) gene. Time-dependent histopathological, immunohistochemical, and gene expression analyses were performed. The ACLT + climbing group showed more severe cartilage defects and massive osteophyte formation than the ACLT group. Smo deletion significantly suppressed PTOA progression. The time-dependent assessment revealed cartilaginous processes of equivalent size at the posterior tibial margin in the Smo cKO and control mice at 4 weeks postoperatively. However, at 8 weeks postoperatively, mature ossifying lesions were detected in the controls but not in Smo cKO mice. In the articular cartilage, ADAMTS5 and RUNX2 expression were observed in hypertrophic chondrocytes near the defective cartilage in controls but not in Smo cKO mice. Climbing exercise after ACLT accelerated PTOA progression more severely not only through increasing joint instability induced by ACLT but also through mechanical loading force induced by climbing exercise. Hedgehog signaling inhibition attenuated PTOA progression by suppressing chondrocyte hypertrophy induced by mechanical loads, to which ACL-deficient athletes are usually exposed. Thus, Hedgehog signaling inhibition may be a therapeutic option to prevent arthritic changes in athletes. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:609-619, 2020.

MEMRI-based imaging pipeline for guiding preclinical studies in mouse models of sporadic medulloblastoma.

Genetically engineered mouse models of sporadic cancers are critical for studying tumor biology and for preclinical testing of therapeutics. We present an MRI-based pipeline designed to produce high resolution, quantitative information about tumor progression and response to novel therapies in mouse models of medulloblastoma (MB). Sporadic MB was modeled in mice by inducing expression of an activated form of the Smoothened gene (aSmo) in a small number of cerebellar granule cell precursors. aSmo mice were imaged and analyzed at defined time-points using a 3D manganese-enhanced MRI-based pipeline optimized for high-throughput. A semi-automated segmentation protocol was established that estimates tumor volume in a time-frame compatible with a high-throughput pipeline. Both an empirical, volume-based classifier and a linear discriminant analysis-based classifier were tested to distinguish progressing from nonprogressing lesions at early stages of tumorigenesis. Tumor centroids measured at early stages revealed that there is a very specific location of the probable origin of the aSmo MB tumors. The efficacy of the manganese-enhanced MRI pipeline was demonstrated with a small-scale experimental drug trial designed to reduce the number of tumor associated macrophages and microglia. Our results revealed a high level of heterogeneity between tumors within and between aSmo MB models, indicating that meaningful studies of sporadic tumor progression and response to therapy could not be conducted without an imaging-based pipeline approach.

Differential inflammatory landscape stimulus during titanium surfaces obtained osteogenic phenotype.

Molecular mechanism governing inflammatory scenario in response to titanium (Ti)-nanotexturing surfaces needs to be better addressed. Thus, we subjected pre-osteoblast to different Ti-texturing surfaces, as follows: machined (Mac), double acid-etching (DAE), and nanoscaled hydroxyapatite-blasted titanium surface (nHA), considering the cells chronically responding either directly (when the cells were cultured onto the surfaces) or indirectly (when the cells were challenged with the conditioned medium by the surfaces), up to 10 days. Our results showed that there is a dynamic requirement of inflammatory-related genes activation in response to nHA by up expressing IL1ß, IL6, IL10, and IL33 (direct condition) and IL6, IL10, IL18 (indirect condition). Importantly, our data show that there is inflammasome involvement, once NLRP3, ASC1, and CASP1 genes were also required. As we found a strong signal of IL10, an anti-inflammatory cytokine, we further investigated Sonic Hedgehog (Shh) signaling cascade. Surprisingly, Shh ligand and Smoothened (Smo) genes were up-modulated in response to nHA, while Patched (Ptc) was down-modulated. Finally, an interactome was built using bioinformatics reinforcing Shh signaling cascade on modulating IL10 transcripts by Src mediating this process and this prevalence of anti-inflammatory picture might explain the low profile of RANKL transcripts in response to nHA, compromising the osteoclastogenesis surrounding the implants. Taking our results into account, our data show that the inflammatory landscape promoted by nHA is strictly modulated by Shh signaling promoted anti-inflammatory pathways. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1597-1604, 2019.

Effects of Smoothened gene targeting small interfering RNA lentiviral vector on biological characteristics of human pancreatic cancer cells

Objective To observe the effect of lentiviral vector carrying Smoothened (SMO) gene targeting small interfering RNA (siRNA) on biological characteristics of pancreatic cancer cells in vitro. Methods Based on previous research methods, 3 lentiviral vectors with SMO siRNA (titer: 5×108, 6×108, 5×108 TU/ml) were synthesized and transfected into Patu-8988 cells. The expression of SMO gene in cells was detected by real-time quantitative polymerase chain reaction (Real-time PCR), and the inhibition rate of gene expression was calculated. Interference vectors with the highest inhibition rate were screened to transfect into Patu-8988 for 72 h. Cell growth was detected by methyl thiazol tetrazolium (MTT), cell apoptosis by flow cytometry, cell invasion and metastasis ability by Transwell invasion assay, and the expression of pathway target gene B cell lymphoma/leukemia-2 (bcl-2) by Western blotting, respectively. Results The inhibition rate of 3 lentiviral vectors of SMO siRNA on SMO gene expression in Patu-8988 cells was 82.4%, 93.1% and 89.1%, respectively. With the selection of interference vector with the highest inhibition rate, it was transfected into Patu-8988 cells for 72 h, and cells grew well. As compared with the negative control group, the growth inhibition rate of Patu-8988 cells in transfected group was (7.46±0.32)%, (25.41±0.28)%, (25.94±0.51)%, (26.01±0.94)% and (43.23±0.88)% from the first to the fifth day, respectively; the apoptotic rate was (1.35±0.11)%, which was lower than that of the control group in which no significant apoptosis was observed (t=-4.523, P=0.011). The number of cells observed in the Invasion chamber and Transwell chamber was (38.00±4.73)/HP and (78.00±3.71)/HP in the transfected group, respectively, significantly lower than those in the control group (128.00±10.91)/HP and (306.00±9.12)/HP, respectively (t=-22.808, P=0.000; t=-69.256, P=0.000); the relative expression of bcl-2 protein was down-regulated in the cells (t=-27.386, P=0.000). Conclusion SMO small interfering RNA lentiviral vector can effectively block the Hedgehog signaling pathway, and significantly inhibit the growth, invasion and metastasis of Patu-8988 cells. Key words: Pancreatic carcinoma; Hedgehog signal transduction pathway; Smoothened gene; Small interfering RNA

Abstract 2435: Smoothened a new perspective for uterine leiomyoma treatment

Abstract Background: Uterine leiomyomas (LM) are the most common gynecologic tumors occurring in women of reproductive age. Patients may present clinical complications such as bleeding, pain and infertility. Additionally, some researchers believe that a leiomyosarcoma can arise from a degenerated LM. Efficient treatments for LM are still limited. Identification of gene markers can help to elucidate these tumors pathogenesis and to reveal molecules that can be used as therapeutic targets. Objective: To assess gene and miRNAs expression, and DNA methylation profiles of genes related to the Sonic Hedgehog (SHH) signaling pathway in uterine leiomyoma samples in order to look for potential therapeutic targets. Methods: 80 samples of LM and 20 myometrium (MM) were obtained in the Department of Obstetrics and Gynecology at HCFMUSP/Sao Paulo - Brazil. Expression profile of 106 genes and 84 miRNAs sequences related to SHH regulation, and the percentage of methylation in the promoter region of the SHH, PTCH1, SMO, SUFU, GLI and GLI3 genes were performed by quantitative Real Time PCR. Results: Among 106 genes evaluated, 16 showed hyper- and 11 showed hypoexpression in LM samples, compared to MM. Only SMOOTHENED (SMO) was found hyperegulated in LM. miRNAs analysis showed 16 sequences with different regulation profiles between LM and MM. None of them is involved in the regulation of SMO expression. DNA methylation profile showed a higher percentage of methylation in LM samples, but this difference did not exceed 2% of methylation for the PTCH1, SMO, GLI1 and GLI3 genes. Conclusion: Our results suggest an activation of SHH pathway in LMs by hyperexpression of SMO. These results are very interesting because there are SMO specific drugs approved by FDA for other tumors types. Citation Format: Natalia Garcia, Laura Gonzalez Anjos, Giovana De Nardo Maffazioli, Nilo Bozzini, Edmund Chada Baracat, Katia Candido Carvalho. Smoothened a new perspective for uterine leiomyoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2435. doi:10.1158/1538-7445.AM2017-2435

Response of germline and somatic smoothened (SMO) mutations in non-small cell lung cancer (NSCLC) to hedgehog inhibitor vismodegib.

9062 Background: Smoothened (SMO) gene somatic mutations activate hedgehog signaling in basal cell cancers (BCC) and medulloblastomas but have not been reported in NSCLC. We detected somatic and germline SMOmutations in NSCLC patients (A-C) and sought to characterize the mutations further. Methods: We performed tumor/blood germline sequencing of SMO mutations, familial germline mutation testing (saliva specimens), somatic mutation analysis of TCGA lung carcinoma datasets, and germline mutation analysis of the TCGA and 3 additional large cohorts (n = 1933). To evaluate the functional significance of SMOP641A, HCC4011 lung cancer cells were transfected with a wild-type SMO or SMOP641A expression vector and a predictive model was created. Results: NSCLC SMOP641A in vitro activated the hedgehog pathway, and vismodegib/cyclopamine inhibited tumor cell growth. Structural modeling suggests that SMO P641A induces conformational changes and disrupts PTCH-SMO interaction leading to constitutive activation. In the NSCLC TCGA databases, somatic SMO mutations occur 1.7%. In the overall TCGA database, germline SMO P641A occurred in 0.11% of cancer patients (multiple cancers) compared with 0% in cancer-free individuals. Patient A (never-smoking SCC) had a 46% RECIST reduction within 6 weeks for 6 months on vismodegib. His 3-generation family pedigree identified germline SMOP641A in one daughter (who developed BCC early). Two additional NSCLC patients (B – germline P641A and C –M525L received vismodegib; B initially stabilized but stopped vismodegib after 14 weeks for toxicity while patient C had no response. Conclusions: SMO mutations are targetable, potentially heritable, oncogenic drivers in NSCLC and other cancers. Tumor genetic profiling should consider including SMO gene, especially in never-smoking lung SCC patients. Additional studies are needed to define the role of germline/somatic SMO alterations in promoting carcinogenesis, interactions with P53 alterations, and the responsiveness of different SMO mutations to hedgehog inhibitors. Currently, the ongoing ECOG-ACRIN MATCH study (NCT02465060) treats SMO/PTCH mutated patients with vismodegib.

Hypoxia but not normoxia promotes Smoothened transcription through upregulation of RBPJ and Mastermind-like 3 in pancreatic cancer

We previously demonstrated that Hedgehog (Hh) signaling is activated under hypoxia through upregulation of transcription of Smoothened (SMO) gene. However, the mechanism of hypoxia-induced activation of SMO transcription remains unclear. In the analysis of altered expressions of genes related to Hh signaling between under normoxia and hypoxia by DNA microarray analysis, we picked up 2 genes, a transcriptional regulator, recombination signal binding protein for immunoglobulin-kappa-J region (RBPJ) and a transcriptional co-activator, Mastermind-like 3 (MAML3). Expressions of SMO, MAML3 and RBPJ were increased under hypoxia in pancreatic ductal adenocarcinoma cells (PDAC). RBPJ and MAML3 inhibition under hypoxia led to decreased SMO and GLI1 expressions, whereas SMO expression in MAML3-inhibited and RBPJ-inhibited cells under normoxia showed no change. However, overexpression of RBPJ under normoxia led to increased SMO expression. Additionally, cells knocked down for MAML3 and RBPJ inhibition under hypoxia showed decreased invasiveness through matrix metalloproteinase-2 suppression and decreased proliferation. Xenograft mouse models showed that MAML3 and RBPJ knockdown inhibited tumorigenicity and tumor volume. Our results suggest that hypoxia promotes SMO transcription through upregulation of MAML3 and RBPJ to induce proliferation, invasiveness and tumorigenesis in pancreatic cancer.

Association between donor and recipient smoothened gene polymorphisms and the risk of hepatocellular carcinoma recurrence following orthotopic liver transplantation in a Han Chinese population.

Hepatocellular carcinoma (HCC) recurrence after orthotopic liver transplantation (OLT) is potential cause for the poor outcome. Smoothened (SMO) gene has been considered associating with HCC and HCC recurrence, but its association with HCC recurrence after OLT is not clear yet. In this study, we aim at evaluating the association between donor and recipient SMO gene polymorphisms and HCC recurrence after OLT. A total of 76 patients with HCC who had undergone OLT from July 2007 to August 2012 were included. A single nucleotide polymorphism (SNP), SMO rs3824, located at the 3'UTR region, was genotyped and analyzed in both donor and recipient. We demonstrated that recipient rs3824 polymorphism was significantly associated with HCC recurrence following OLT. In multivariate logistic regression analysis, TNM stage (p = 0.001), recipient SMO rs3824 genotype (CG vs. CC/GG p = 0.001), and histologic grade (p = 0.019) were identified as independent risk factors of HCC recurrence. Recurrence-free survival (RFS) and overall survival (OS) were significantly higher in the recipient CC/GG group than in the CG group (p = 0.003 and p = 0.011, respectively). Cox proportional hazards modeling revealed that TNM stage, recipient SMO rs3824 genotype, pre-OLT serum AFP level, and histologic grade were independent factors (p < 0.05) for patients' clinical outcomes. In conclusion, recipient SMO rs3824 polymorphism is associated with an increased risk of HCC recurrence following OLT and has a potential clinical value for the prognosis of HCC patients treated with OLT.

A role for smoothened during murine lens and cornea development.

Various studies suggest that Hedgehog (Hh) signalling plays roles in human and zebrafish ocular development. Recent studies (Kerr et al., Invest Ophthalmol Vis Sci. 2012; 53, 3316–30) showed that conditionally activating Hh signals promotes murine lens epithelial cell proliferation and disrupts fibre differentiation. In this study we examined the expression of the Hh pathway and the requirement for the Smoothened gene in murine lens development. Expression of Hh pathway components in developing lens was examined by RT-PCR, immunofluorescence and in situ hybridisation. The requirement of Smo in lens development was determined by conditional loss-of-function mutations, using LeCre and MLR10 Cre transgenic mice. The phenotype of mutant mice was examined by immunofluorescence for various markers of cell cycle, lens and cornea differentiation. Hh pathway components (Ptch1, Smo, Gli2, Gli3) were detected in lens epithelium from E12.5. Gli2 was particularly localised to mitotic nuclei and, at E13.5, Gli3 exhibited a shift from cytosol to nucleus, suggesting distinct roles for these transcription factors. Conditional deletion of Smo, from ∼E12.5 (MLR10 Cre) did not affect ocular development, whereas deletion from ∼E9.5 (LeCre) resulted in lens and corneal defects from E14.5. Mutant lenses were smaller and showed normal expression of p57Kip2, c-Maf, E-cadherin and Pax6, reduced expression of FoxE3 and Ptch1 and decreased nuclear Hes1. There was normal G1-S phase but decreased G2-M phase transition at E16.5 and epithelial cell death from E14.5-E16.5. Mutant corneas were thicker due to aberrant migration of Nrp2+ cells from the extraocular mesenchyme, resulting in delayed corneal endothelial but normal epithelial differentiation. These results indicate the Hh pathway is required during a discrete period (E9.5–E12.5) in lens development to regulate lens epithelial cell proliferation, survival and FoxE3 expression. Defective corneal development occurs secondary to defects in lens and appears to be due to defective migration of peri-ocular Nrp2+ neural crest/mesenchymal cells.

Smoothened gene alterations in keratocystic odontogenic tumors.

BackgroundIt has been widely demonstrated that the hedgehog pathway is strongly associated with basal cell carcinoma of the skin (NBCCS). To assess potential DNA alterations related to keratocystic odontogenic tumors (KCOTs), we sequenced smoothened (SMO) genes in 12 sporadic KCOTs.MethodsPolymerase chain reaction (PCR), capillary electrophoresis and dideoxy chain-termination sequencing were used to examine potential DNA alterations in sporadic KCOTs.ResultsFive alterations in SMO genes were detected. Four of these mutations consisted of two synonymous and three missense mutations; two of which have not been reported to date (c.T776A, c.T1281G).ConclusionsSMO genes may play an important role in the sonic hedgehog (SHH) pathway and could also be responsible for generating KCOTs and NBCCS. However, their influence on SHH signaling remains to be elucidated.

16IN - Meningioma: what Can we Learn from the Biology for Precision Medicine?

ABSTRACT Meningiomas differ considerably by their histological appearance, location, biological behavior, recurrence rate, and overall survival. The most frequent alteration found in sporadic meningioma corresponds to biallelic NF2 inactivation. Besides the central role of NF2, other alternative genetic alterations have been recently identified in meningioma initiation. The AKT1E17K mutation was found in 12%, predominantly in benign skull base meningiomas, occurring frequently together with mutations in the TRAF7 gene. TRAF7 mutations are exclusively of NF2 mutations and occur in about 24% of meningiomas. In addition, meningiomas with TRAF7 mutations are almost always characterized by recurrent mutation in the KLF4 gene (co-regulator of the bradykinin B2 receptor). Another non-NF2-associated mutation of meningiomas affects the SMO (Smoothened) gene which is a member of the hedgehog pathway. SMO mutations occur in 5% of grade I meningiomas and are restricted to the midline skull base. Meningioma progression and recurrence are linked to chromosome instability, CDKN2AB genes on chromosome 9p being suspected to be important. Mouse models that reproduce human meningioma tumorigenesis have been developed. Two conditional Nf2 knockout models have shown that Nf2 inactivation in leptomeningeal cells of mice is sufficient to induce meningiomas. Additional deletion of Cdkn2ab, together with Nf2 inactivation, results in increased meningioma frequency, as well as development of grade II or grade III meningiomas. All relevant growth factor receptors/kinases have been described to be expressed in meningiomas, including epidermal growth factor receptor (EGFR), platelet-derived growth factor beta receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and insulin-like growth factor receptor (IGFR). A new mouse model surexpressing PDGF-beta has been recently developed showing an increase in frequency and malignancy in association with Nf2 and Cdkn2ab inactivation. In conclusion, increasing knowledge on the molecular mechanisms of meningioma initiation and development provides target that could be relevant to develop new therapeutic approaches, particularly in surgery and radiotherapy-refractory meningiomas. Disclosure: All authors have declared no conflicts of interest.

Targeting the SMO oncogene by miR-326 inhibits glioma biological behaviors and stemness.

Few studies have associated microRNAs (miRNAs) with the hedgehog (Hh) pathway. Here, we investigated whether targeting smoothened (SMO) with miR-326 would affect glioma biological behavior and stemness. To investigate the expression of SMO and miR-326 in glioma specimens and cell lines, we utilized quantitative real-time (qRT)-PCR, Western blot, immunohistochemistry, and fluorescence in situ hybridization. The luciferase reporter assay was used to verify the relationship between SMO and miR-326. We performed cell counting kit-8, transwell, and flow cytometric assays using annexin-V labeling to detect changes after transfection with siRNA against SMO or miR-326. qRT-PCR assays, neurosphere formation, and immunofluorescence were utilized to detect the modification of self-renewal and stemness in U251 tumor stem cells. A U251-implanted intracranial model was used to study the effect of miR-326 on tumor volume and SMO suppression efficacy. SMO was upregulated in gliomas and was associated with tumor grade and survival period. SMO inhibition suppressed the biological behaviors of glioma cells. SMO expression was inversely correlated with miR-326 and was identified as a novel direct target of miR-326. miR-326 overexpression not only repressed SMO and downstream genes but also decreased the activity of the Hh pathway. Moreover, miR-326 overexpression decreased self-renewal and stemness and partially prompted differentiation in U251 tumor stem cells. In turn, the inhibition of Hh partially elevated miR-326 expression. Intracranial tumorigenicity induced by the transfection of miR-326 was reduced and was partially mediated by the decreased SMO expression. This work suggests a possible molecular mechanism of the miR- 326/SMO axis, which can be a potential alternative therapeutic pathway for gliomas.