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BACKGROUND: Autophagy may be involved in the pathological process of steroid-induced necrosis of the femoral head (SINFH). Some studies have confirmedthat circular RNAs (circRNAs) have a regulatory mechanism in SINFH; however, whether circCDR1as affects autophagy in SINFH has not been investigated. OBJECTIVE: To explore the level of autophagy and the regulatory mechanism of circCDR1as in SINFH. METHODS: Gene expression profiles of SINFH and control rats were extracted from the GSE26316 dataset and differential expression analysis was performed. Subsequently, the biological functions of differentially expressed genes were analyzed. Then, the target miRNAs of circCDR1as and the target genes of target miRNAs were predicted. Further, the target genes were compared with the differentially expressed genes to construct the regulatory network of circCDR1as. In addition, femoral head samples from patients with SINFH and healthy control individuals were collected. Bone marrow mesenchymal stem cells were also applied for cellular experiments and randomly divided into bone marrow mesenchymal stem cell group, model group (methylprednisolone-treated), model+siNC group, and model+si-CDR1as group. RT-qPCR was used to detect the expression of circCDR1as and target genes in cells and tissue samples. Western blot was used to examine the expression of autophagy proteins. The luciferase reporter gene vectors, pmirGLO-CDR1as (WT), pmirGLO-RAF1 (WT), pmirGLOCDR1as (MUT), and pmirGLO-RAF1 (MUT), were constructed and transfected into the cells. miR-7-5p mimic and mimic NC groups were established. The targetregulatory relationship of the circCDR1as network was detected. RESULTS AND CONCLUSION: A total of 1 283 differentially expressed genes were identified between the SINFH and control groups, which were mainly involved in apoptotic and autophagic signaling pathways. Prediction analysis revealed that circCDR1as targeted 6 miRNAs, which in turn regulated 305 target genes. Among these target genes, 31 showed differential expression in SINFH. Among the differentially expressed target genes, RAF1, involved in autophagy, was selected as a key gene, leading to the construction of the circCDR1as/miR-7-5p/RAF1 regulatory network. Compared with the control group, circCDR1as, RAF1, and autophagy levels were upregulated in patients with SINFH and in hormone-induced bone marrow mesenchymal stem cells (P < 0.05), while miR-7-5p expression was downregulated (P < 0.05). Knockdown of circCDR1as significantly decreased cellular autophagy levels (P < 0.05). Dual-luciferase reporter assays confirmed the targeting relationships between circCDR1as and miR-7-5p, as well as between miR-7-5p and RAF1. To conclude, the CircCDR1as/miR-7-5p/RAF1 may potentially promote SINFH through autophagy. Targeting circCDR1as is a potential strategy for partial autophagic repair in the treatment of SINFH. [ABSTRACT FROM AUTHOR]

Epilepsy is a common neurological disorder, and the exploration of potential therapeutic drugs for its treatment is still ongoing. Vitamin D has emerged as a promising treatment due to its potential neuroprotective effects and anti-epileptic properties. This study aimed to investigate the effects of vitamin D on epilepsy and neuroinflammation in juvenile mice using network pharmacology and molecular docking, with a focus on the mammalian target of rapamycin (mTOR) signaling pathway. Experimental mouse models of epilepsy were established through intraperitoneal injection of pilocarpine, and in vitro injury models of hippocampal neurons were induced by glutamate (Glu) stimulation. The anti-epileptic effects of vitamin D were evaluated both in vivo and in vitro. Network pharmacology and molecular docking analysis were used to identify potential targets and regulatory pathways of vitamin D in epilepsy. The involvement of the mTOR signaling pathway in the regulation of mouse epilepsy by vitamin D was validated using rapamycin (RAPA). The levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were assessed by enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining was used to analyze the apoptosis of hippocampal neurons. In in vivo experiments, vitamin D reduced the Racine scores of epileptic mice, prolonged the latency of epilepsy, and inhibited the production of TNF-α, IL-1β, and IL-6 in the hippocampus. Furthermore, network pharmacology analysis identified RAF1 as a potential target of vitamin D in epilepsy, which was further confirmed by molecular docking analysis. Additionally, the mTOR signaling pathway was found to be involved in the regulation of mouse epilepsy by vitamin D. In in vitro experiments, Glu stimulation upregulated the expressions of RAF1 and LC3II/LC3I, inhibited mTOR phosphorylation, and induced neuronal apoptosis. Mechanistically, vitamin D activated the mTOR signaling pathway and alleviated mouse epilepsy via RAF1, while the use of the pathway inhibitor RAPA reversed this effect. Vitamin D alleviated epilepsy symptoms and neuroinflammation in juvenile mice by activating the mTOR signaling pathway via RAF1. These findings provided new insights into the molecular mechanisms underlying the anti-epileptic effects of vitamin D and further supported its use as an adjunctive therapy for existing anti-epileptic drugs. [ABSTRACT FROM AUTHOR]

Introduction: Here, we report the first case of a soft tissue tumor with a PAK2::RAF1 fusion. Case Presentation: The patient was an 11-year-old female presenting with a 5 cm intramuscular mass in the lower leg. Microscopic examination revealed a mitotically active spindle cell lesion with monomorphic and moderately atypical cells growing in a patternless pattern with the presence of stromal and perivascular keloidal collagen with focal immunoreactivity for smooth muscle actin and S100; negative stains included cytokeratins, CD34, and caldesmon. Whole genome and RNA sequencing detected a chromosome 3 inversion and a resultant PAK2::RAF1 fusion as well as a CDKN2A homozygous deletion. The patient was treated with neoadjuvant chemoradiotherapy with only minimal response and the tumor was excised surgically. There was no evidence of disease progression at 4 months. Conclusion: This is the first case of a soft tissue tumor harboring a PAK2::RAF1 fusion with histological features in keeping with previous cases of RAF1 and other kinase fusion soft tissue tumors. [ABSTRACT FROM AUTHOR]

Background: Sterigmatocystin (STE) is a mycotoxin widely found in contaminated food and foodstuffs, and excessive long-term exposure to STE is associated with several health issues, including infertility. However, there is little information available regarding the effects of STE toxin on the female reproductive system, particularly concerning oocyte maturation. Methods: In the present study, we investigated the toxic effects of STE on mouse oocyte maturation. We also used Western blot, immunofluorescence, and image quantification analyses to assess the impact of STE exposure on the oocyte maturation progression, mitochondrial distribution, oxidative stress, DNA damages, oocyte ferroptosis and asymmetric division defects. Results: Our results revealed that STE exposure disrupted mouse oocyte maturation progression. When we examined the cellular changes following 100 µM STE treatment, we found that STE adversely affected polar body extrusion and induced asymmetric division defects in oocytes. RNA-sequencing data showed that STE exposure affects the expression of several pathway-correlated genes during oocyte meiosis in mice, suggesting its toxicity to oocytes. Based on the RNA-seq data, we showed that STE exposure induced oxidative stress and caused DNA damage in oocytes. Besides, ferroptosis and α-tubulin acetylation were also found in STE-exposed oocytes. Moreover, we determined that STE exposure resulted in reduced RAF1 protein expression in mouse oocytes, and inhibition of RAF1 activity also causes defects in asymmetric division of mouse oocytes. Conclusions: Collectively, our research provides novel insights into the molecular mechanisms whereby STE contributes to abnormal meiosis. [ABSTRACT FROM AUTHOR]

Introduction: Here, we report the first case of a soft tissue tumor with a PAK2::RAF1 fusion. Case Presentation: The patient was an 11-year-old female presenting with a 5 cm intramuscular mass in the lower leg. Microscopic examination revealed a mitotically active spindle cell lesion with monomorphic and moderately atypical cells growing in a patternless pattern with the presence of stromal and perivascular keloidal collagen with focal immunoreactivity for smooth muscle actin and S100; negative stains included cytokeratins, CD34, and caldesmon. Whole genome and RNA sequencing detected a chromosome 3 inversion and a resultant PAK2::RAF1 fusion as well as a CDKN2A homozygous deletion. The patient was treated with neoadjuvant chemoradiotherapy with only minimal response and the tumor was excised surgically. There was no evidence of disease progression at 4 months. Conclusion: This is the first case of a soft tissue tumor harboring a PAK2::RAF1 fusion with histological features in keeping with previous cases of RAF1 and other kinase fusion soft tissue tumors.

Abstract Background Sterigmatocystin (STE) is a mycotoxin widely found in contaminated food and foodstuffs, and excessive long-term exposure to STE is associated with several health issues, including infertility. However, there is little information available regarding the effects of STE toxin on the female reproductive system, particularly concerning oocyte maturation. Methods In the present study, we investigated the toxic effects of STE on mouse oocyte maturation. We also used Western blot, immunofluorescence, and image quantification analyses to assess the impact of STE exposure on the oocyte maturation progression, mitochondrial distribution, oxidative stress, DNA damages, oocyte ferroptosis and asymmetric division defects. Results Our results revealed that STE exposure disrupted mouse oocyte maturation progression. When we examined the cellular changes following 100 µM STE treatment, we found that STE adversely affected polar body extrusion and induced asymmetric division defects in oocytes. RNA-sequencing data showed that STE exposure affects the expression of several pathway-correlated genes during oocyte meiosis in mice, suggesting its toxicity to oocytes. Based on the RNA-seq data, we showed that STE exposure induced oxidative stress and caused DNA damage in oocytes. Besides, ferroptosis and α-tubulin acetylation were also found in STE-exposed oocytes. Moreover, we determined that STE exposure resulted in reduced RAF1 protein expression in mouse oocytes, and inhibition of RAF1 activity also causes defects in asymmetric division of mouse oocytes. Conclusions Collectively, our research provides novel insights into the molecular mechanisms whereby STE contributes to abnormal meiosis.

We report an exceptional case of a spindle cell mesenchymal tumor with S100 and CD34 co-reactivity, which harbored a SLMAP::RAF1 fusion. To the best of our knowledge, this is the second case of a spindle cell mesenchymal tumor with S100 and CD34 co-reactivity with this specific fusion. Remarkable is the presence of calcification and heterotopic ossification in the center of our lesion, a feature that, to our knowledge, has not been described yet in RAF1 -rearranged spindle cell mesenchymal tumors. [ABSTRACT FROM AUTHOR]

Colorectal Carcinoma (CRC) is one of the most common malignant tumors of the digestive tract, with a high mortality rate. DPY30 is one of the core subunits of the histone methyltransferase complex, which was involved in many cancer processes. However, the role of DPY30 in the occurrence and progression of CRC remains unclear. In this study, we sought to evaluate the role and mechanism of DPY30 in CRC cells apoptosis. Here, we identified that knockdown of DPY30 significantly inhibited the HT29 and HCT116 cells proliferation in vitro. Moreover, the knockdown of DPY30 significantly increased the apoptosis rate and promoted the expression of apoptosis-related proteins in CRC cells. Meanwhile, DPY30 knockdown promoted CRC cells apoptosis through endogenous programmed death and in a caspase activation-dependent manner. Furthermore, RNA-seq analysis revealed that the action of DPY30 is closely related to the apoptosis biological processes, and screened its potential effectors Raf1. Mechanistically, DPY30 downregulation promotes MST2-induced apoptosis by inhibiting Raf1 transcriptional activity through histone H3 lysine 4 trimethylation (H3K4me3). In vivo experiments showed that DPY30 was correlated with Raf1 in nude mouse subcutaneous xenografts tissues significantly. Clinical colorectal specimens further confirmed that overexpression of DPY30 in malignant tissues was significantly correlated with Raf1 level. The vital role of the DPY30/Raf1/MST2 signaling axis in the cell death and survival rate of CRC cells was disclosed, which provides potential new targets for early diagnosis and clinical treatment of CRC.

Abstract Background Endometrial carcinoma (EC) is one of the world’s typical female reproductive tract malignancies, mostly occurring in postmenopausal women. Many reports have confirmed that long non-coding RNA SOX21 antisense RNA1 (lncRNA SOX21-AS1) is associated with the progressions of various cancer. However, the mechanism of SOX21-AS1 in EC remains unclear. Our study is intended to probe the mechanisms of SOX21-AS1 on EC progression. Methods The CCK-8 assay and colony formation detected cell proliferation. Cell migration and invasion were assessed by transwell analysis. Apoptosis was measured by flow cytometry assay. Bioinformatics software predicted target binding and confirmed using a luciferase reporter analysis. Results SOX21-AS1 expression was upregulated in EC tumor tissues and cells. High expression of SOX21-AS1 was associated with poor overall survival. Silencing of SOX21-AS1 restrained cell proliferation, migration, invasion, and increased apoptosis in HEC-1A and Ishikawa cells. Additionally, bioinformatics analysis demonstrated that SOX21-AS1 modulated RAF1 expression by competitively binding to miR-7-5p. Functionally, silencing of RAF1 reversed the functions of miR-7-5p inhibitor in the proliferation, invasion, and apoptosis of HEC-1A/sh-SOX21-AS1 and Ishikawa/sh-SOX21-AS1 cells. Conclusions SOX21-AS1 promoted the pathological development of EC by regulating the miR-7-5p/RAF1 pathway. This research may provide a novel target for EC therapy.

Abstract The dimerization of RAF kinases represents a key event in their activation cycle and in RAS/ERK pathway activation. Genetic, biochemical and structural approaches provided key insights into this process defining RAF signaling output and the clinical efficacy of RAF inhibitors (RAFi). However, methods reporting the dynamics of RAF dimerization in living cells and in real time are still in their infancy. Recently, split luciferase systems have been developed for the detection of protein–protein-interactions (PPIs), incl. proof-of-concept studies demonstrating the heterodimerization of the BRAF and RAF1 isoforms. Due to their small size, the Nanoluc luciferase moieties LgBiT and SmBiT, which reconstitute a light emitting holoenzyme upon fusion partner promoted interaction, appear as well-suited to study RAF dimerization. Here, we provide an extensive analysis of the suitability of the Nanoluc system to study the homo- and heterodimerization of BRAF, RAF1 and the related KSR1 pseudokinase. We show that KRASG12V promotes the homo- and heterodimerization of BRAF, while considerable KSR1 homo- and KSR1/BRAF heterodimerization already occurs in the absence of this active GTPase and requires a salt bridge between the CC-SAM domain of KSR1 and the BRAF-specific region. We demonstrate that loss-of-function mutations impairing key steps of the RAF activation cycle can be used as calibrators to gauge the dynamics of heterodimerization. This approach identified the RAS-binding domains and the C-terminal 14–3-3 binding motifs as particularly critical for the reconstitution of RAF mediated LgBiT/SmBiT reconstitution, while the dimer interface was less important for dimerization but essential for downstream signaling. We show for the first time that BRAFV600E, the most common BRAF oncoprotein whose dimerization status is controversially portrayed in the literature, forms homodimers in living cells more efficiently than its wildtype counterpart. Of note, Nanoluc activity reconstituted by BRAFV600E homodimers is highly sensitive to the paradox-breaking RAFi PLX8394, indicating a dynamic and specific PPI. We report the effects of eleven ERK pathway inhibitors on RAF dimerization, incl. third-generation compounds that are less-defined in terms of their dimer promoting abilities. We identify Naporafenib as a potent and long-lasting dimerizer and show that the split Nanoluc approach discriminates between type I, I1/2 and II RAFi. Video Abstract

Abstract RAF1 (c‐raf) has been known as an important tumor‐promoter in many cancers. However, the regulatory mechanisms affecting RAF1 expressions are rarely reported. This study aimed to predict the candidate miRNAs of RAF1 gene and verify their functions in the progression of lung cancer. The expression of miR‐643 was detected by reverse transcription polymerase chain reaction (RT‐PCR). A dual‐luciferase report system was used to verify the relationship between miR‐643 and RAF1. RT‐PCR and Western blot were used to analyze the regulatory relationship between miR‐643 and RAF1. Transwell chamber, scratch, and monoclonal tests showed that miR‐643 affected the proliferation, migration, and radiosensitivity of H1299 and A549 cells by targeting the RAF1 gene. The expression of miR‐643 in lung cancer cells was lower than that in the bronchial epithelioid cells (CRL‐2741), and luciferase reporter experiment confirmed that miR‐643 targeted RAF1. MiR‐643 overexpression decreased the RAF1 expression, thereby decreasing cell migration, proliferation, and radiation resistance through the AKT/nuclear factor‐κB pathway. miR‐643 in lung cancer cells could inhibit cell proliferation, invasion, and metastasis and increase the radiosensitivity by downregulating RAF1.

Fusions involving CRAF (RAF1) are infrequent oncogenic drivers in pediatric low‐grade gliomas, rarely identified in tumors bearing features of pilocytic astrocytoma, and involving a limited number of known fusion partners. We describe recurrent TRAK1::RAF1 fusions, previously unreported in brain tumors, in three pediatric patients with low‐grade glial‐glioneuronal tumors. We present the associated clinical, histopathologic and molecular features. Patients were all female, aged 8 years, 15 months, and 10 months at diagnosis. All tumors were located in the cerebral hemispheres and predominantly cortical, with leptomeningeal involvement in 2/3 patients. Similar to previously described activating RAF1 fusions, the breakpoints in RAF1 all occurred 5′ of the kinase domain, while the breakpoints in the 3′ partner preserved the N‐terminal kinesin‐interacting domain and coiled‐coil motifs of TRAK1. Two of the three cases demonstrated methylation profiles (v12.5) compatible with desmoplastic infantile ganglioglioma (DIG)/desmoplastic infantile astrocytoma (DIA) and have remained clinically stable and without disease progression/recurrence after resection. The remaining tumor was non‐classifiable; with focal recurrence 14 months after initial resection; the patient remains symptom free and without further recurrence/progression (5 months post re‐resection and 19 months from initial diagnosis). Our report expands the landscape of oncogenic RAF1 fusions in pediatric gliomas, which will help to further refine tumor classification and guide management of patients with these alterations. [ABSTRACT FROM AUTHOR]

Background: Endometrial carcinoma (EC) is one of the world's typical female reproductive tract malignancies, mostly occurring in postmenopausal women. Many reports have confirmed that long non-coding RNA SOX21 antisense RNA1 (lncRNA SOX21-AS1) is associated with the progressions of various cancer. However, the mechanism of SOX21-AS1 in EC remains unclear. Our study is intended to probe the mechanisms of SOX21-AS1 on EC progression. Methods: The CCK-8 assay and colony formation detected cell proliferation. Cell migration and invasion were assessed by transwell analysis. Apoptosis was measured by flow cytometry assay. Bioinformatics software predicted target binding and confirmed using a luciferase reporter analysis. Results: SOX21-AS1 expression was upregulated in EC tumor tissues and cells. High expression of SOX21-AS1 was associated with poor overall survival. Silencing of SOX21-AS1 restrained cell proliferation, migration, invasion, and increased apoptosis in HEC-1A and Ishikawa cells. Additionally, bioinformatics analysis demonstrated that SOX21-AS1 modulated RAF1 expression by competitively binding to miR-7-5p. Functionally, silencing of RAF1 reversed the functions of miR-7-5p inhibitor in the proliferation, invasion, and apoptosis of HEC-1A/sh-SOX21-AS1 and Ishikawa/sh-SOX21-AS1 cells. Conclusions: SOX21-AS1 promoted the pathological development of EC by regulating the miR-7-5p/RAF1 pathway. This research may provide a novel target for EC therapy. [ABSTRACT FROM AUTHOR]

The dimerization of RAF kinases represents a key event in their activation cycle and in RAS/ERK pathway activation. Genetic, biochemical and structural approaches provided key insights into this process defining RAF signaling output and the clinical efficacy of RAF inhibitors (RAFi). However, methods reporting the dynamics of RAF dimerization in living cells and in real time are still in their infancy. Recently, split luciferase systems have been developed for the detection of protein–protein-interactions (PPIs), incl. proof-of-concept studies demonstrating the heterodimerization of the BRAF and RAF1 isoforms. Due to their small size, the Nanoluc luciferase moieties LgBiT and SmBiT, which reconstitute a light emitting holoenzyme upon fusion partner promoted interaction, appear as well-suited to study RAF dimerization. Here, we provide an extensive analysis of the suitability of the Nanoluc system to study the homo- and heterodimerization of BRAF, RAF1 and the related KSR1 pseudokinase. We show that KRASG12V promotes the homo- and heterodimerization of BRAF, while considerable KSR1 homo- and KSR1/BRAF heterodimerization already occurs in the absence of this active GTPase and requires a salt bridge between the CC-SAM domain of KSR1 and the BRAF-specific region. We demonstrate that loss-of-function mutations impairing key steps of the RAF activation cycle can be used as calibrators to gauge the dynamics of heterodimerization. This approach identified the RAS-binding domains and the C-terminal 14–3-3 binding motifs as particularly critical for the reconstitution of RAF mediated LgBiT/SmBiT reconstitution, while the dimer interface was less important for dimerization but essential for downstream signaling. We show for the first time that BRAFV600E, the most common BRAF oncoprotein whose dimerization status is controversially portrayed in the literature, forms homodimers in living cells more efficiently than its wildtype counterpart. Of note, Nanoluc activity reconstituted by BRAFV600E homodimers is highly sensitive to the paradox-breaking RAFi PLX8394, indicating a dynamic and specific PPI. We report the effects of eleven ERK pathway inhibitors on RAF dimerization, incl. third-generation compounds that are less-defined in terms of their dimer promoting abilities. We identify Naporafenib as a potent and long-lasting dimerizer and show that the split Nanoluc approach discriminates between type I, I1/2 and II RAFi. Ah1kwifp3aoZnF_eXdbERz Video Abstract [ABSTRACT FROM AUTHOR]

Objective: To investigate the therapeutic effect of gentisic acid (GA) on rheumatoid arthritis (RA) based on the miR-19b-3p/RAF1 axis. Methods: The cell counting kit-8 method was used to detect the growth inhibitory effect of different concentrations of GA on MH7A cells, and the drug concentration of GA was determined in the experiment. The quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-19b-3p and RAF1. RAF1, extracellular regulated protein kinases1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) were examined by Western blotting. Three methods (dual-luciferase assay, qRT-PCR and Western blot analysis) were used to verify miR-19b-3p targeting RAF1. Flow cytometry was performed to detect MH7A cell apoptosis. Transwell and wound healing assays were used to determine the invasion and migration capacities of MH7A cells. Results: The growth of MH7A cells was gradually inhibited with increasing GA concentration. When the GA concentration exceeded 80 mmol/L, GA was significantly cytotoxic to MH7A cells, so the half maximal inhibitory concentration of GA for MH7A cells was calculated as 67.019 mmol/L. GA upregulated miR-19b-3p expression, downregulated RAF1 expression, inhibited ERK1/2 phosphorylation, induced MH7A cell apoptosis and suppressed MH7A cell invasion and migration (P<0.05 or P<0.01). RAF1 was identified as the target of miR-19b-3p and reversed inhibitory effects on miR-19b-3p expression (P<0.05 or P<0.01). The miR-19b-3p inhibitor upregulated RAF1 expression and ERK1/2 phosphorylation, suppressed MH7A cell apoptosis and induced MH7A cell invasion and migration (P<0.01). Conclusion: GA regulated miR-19b-3p/RAF1 axis to mediate ERK pathway and inhibit the development of RA. [ABSTRACT FROM AUTHOR]

Somatic and germline gain‐of‐function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor‐prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss‐of‐function of the proto‐oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure‐based prediction and functional tests using human knock‐in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress‐induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis. Synopsis: A novel recessive variant in RAF1 was identified as a possible cause of a hitherto unknown lethal neonatal syndrome with progeroid features. This private p.Thr543Met mutation abolished downstream ERK pathway signaling while increasing susceptibility to apoptosis via ASK1 de‐repression. A homozygous p.Thr543Met mutation in RAF1 was found in a neonate presenting with a syndrome encompassing cutaneous, craniofacial, cardiac and limb anomalies.In cultured cells and in vivo using Xenopus assays the p.Thr543Met mutation behaved as a loss‐of‐function allele that abolishes downstream ERK pathway signaling in response to growth factor stimulation.p.Thr543Met knock‐in in 293T cells revealed that RAF1T543M is unstable and more sensitive to stress‐induced apoptosis via ASK1 de‐repression.These findings describe, for the first time, the consequences of a loss of RAF1 function during human development, contrasting with gain‐of‐function mutations seen in cancer‐prone RASopathies. [ABSTRACT FROM AUTHOR]

Mounting evidence suggests that circular RNAs play important roles in the development and progression of cancers. However, their function in glioblastomas (GBM) is still unclear. By circRNA array analysis, we found that circXPO1 (hsa_circ_102737) was significantly upregulated in GBM, and qPCR analysis verified that the circXPO1 expression level was increased in both GBM tissues and cell lines. Functional studies demonstrated that the knockdown of circXPO1 in GBM cell lines repressed cell proliferation and migration; conversely, the overexpression of circXPO1 promoted the malignancy of GBM cells. In line with these findings, circXPO1 inhibition effectively suppressed gliomagenesis in the in situ transplantation model of nude mice. Through bioinformatic analyses and dual-luciferase reporter assays, we showed that circXPO1 directly bound to miR-7-5p, which acted as a tumor suppressor through the negative regulation of RAF1. In conclusion, our studies suggest that the circXPO1/miR-7-5p/RAF1 axis promotes brain tumor formation and may be a potential therapeutic target for GBM treatment. [ABSTRACT FROM AUTHOR]

Phenotype analysis of the Noonan syndrome (NS) related to RAF1 mutations demonstrates that a high proportion of cases exhibit severe lymphatic dysplasia and congenital heart disease, especially hypertrophic cardiomyopathy. Because of the difficulty of fetal phenotypic assessment, the percentage of cases with multisystemic prenatal presentation as well as the phenotypic variability may be underestimated. We describe a 35 weeks male preterm infant presenting with de novo missense mutation NM_002880.4(RAF1):c.770C>T (p.Ser257Leu), whose death occurred following birth. Antenatal ultrasound showed polyhydramnios, severe ascites, and tongue protrusion. Autopsy revealed multiple congenital anomalies including intrauterine growth restriction, hydrops fetalis, characteristic facial dysmorphia, short and webbed neck, hypertrichosis, severe lungs hypoplasia, thymic hyperplasia, hepato‐splenomegaly, bilateral mild uretero‐hydronephrosis, and mild pontocerebellar hypoplasia. Histology revealed increased hepatic hematopoiesis and iron deposits. This report confirms that NS may be associated with multisystem involvement and provides further evidence for the wide phenotypic variability associated with RAF1 variants. [ABSTRACT FROM AUTHOR]

Abstract Somatic and germline gain‐of‐function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor‐prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss‐of‐function of the proto‐oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure‐based prediction and functional tests using human knock‐in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress‐induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis.

Abstract Background Lymphatic metastasis was an independent prognostic risk factor for hypopharyngeal carcinoma and was the main cause of treatment failure. The purpose of this study was to screen the differential genes and investigate the mechanism of lymphatic metastasis in hypopharyngeal carcinoma. Methods Transcriptome sequencing was performed on primary tumors of patients, and differential genes were screened by bioinformatics analysis. The expression of differential genes was verified by qRT-PCR, western-blotting and immunohistochemical, and prognostic value was analyzed by Kaplan–Meier and log-rank test and Cox’s test. Next, FADU and SCC15 cell lines were used to demonstrate the function of differential genes both in vitro by EdU, Flow cytometry, Wound Healing and Transwell assays and in vivo by a foot-pad xenograft mice model. Proteomic sequencing was performed to screen relevant targets. In addition, in vitro and in vivo experiments were conducted to verify the mechanism of lymphatic metastasis. Results Results of transcriptome sequencing showed that RAF1 was a significantly differential gene in lymphatic metastasis and was an independent prognostic risk factor. In vitro experiments suggested that decreased expression of RAF1 could inhibit proliferation, migration and invasion of tumor cells and promote apoptosis. In vivo experiments indicated that RAF1 could promote tumor growth and lymphatic metastasis. Proteomic sequencing and subsequent experiments suggested that LAGE1 could promote development of tumor and lymphatic metastasis, and was regulated by RAF1. Conclusions It suggests that RAF1 can promote lymphatic metastasis of hypopharyngeal carcinoma by regulating LAGE1, and provide a basis for the exploring of novel therapeutic target and ultimately provide new guidance for the establishment of intelligent diagnosis and precise treatment of hypopharyngeal carcinoma.

Ganglioglioma is the most common epilepsy-associated neoplasm that accounts for approximately 2% of all primary brain tumors. While a subset of gangliogliomas are known to harbor the activating p.V600E mutation in the BRAF oncogene, the genetic alterations responsible for the remainder are largely unknown, as is the spectrum of any additional cooperating gene mutations or copy number alterations. We performed targeted next-generation sequencing that provides comprehensive assessment of mutations, gene fusions, and copy number alterations on a cohort of 40 gangliogliomas. Thirty-six harbored mutations predicted to activate the MAP kinase signaling pathway, including 18 with BRAF p.V600E mutation, 5 with variant BRAF mutation (including 4 cases with novel in-frame insertions at p.R506 in the β3-αC loop of the kinase domain), 4 with BRAF fusion, 2 with KRAS mutation, 1 with RAF1 fusion, 1 with biallelic NF1 mutation, and 5 with FGFR1/2 alterations. Three gangliogliomas with BRAF p.V600E mutation had concurrent CDKN2A homozygous deletion and one additionally harbored a subclonal mutation in PTEN. Otherwise, no additional pathogenic mutations, fusions, amplifications, or deletions were identified in any of the other tumors. Amongst the 4 gangliogliomas without canonical MAP kinase pathway alterations identified, one epilepsy-associated tumor in the temporal lobe of a young child was found to harbor a novel ABL2-GAB2 gene fusion. The underlying genetic alterations did not show significant association with patient age or disease progression/recurrence in this cohort. Together, this study highlights that ganglioglioma is characterized by genetic alterations that activate the MAP kinase pathway, with only a small subset of cases that harbor additional pathogenic alterations such as CDKN2A deletion.

For decades, KRAS mutant lung adenocarcinomas (LUAD) have been refractory to therapeutic strategies based on personalized medicine owing to the complexity of designing inhibitors to selectively target KRAS and downstream targets with acceptable toxicities. The recent development of selective KRASG12C inhibitors represents a landmark after 40 years of intense research efforts since the identification of KRAS as a human oncogene. Here, we discuss the mechanisms responsible for the rapid development of resistance to these inhibitors, as well as potential strategies to overcome this limitation. Other therapeutic strategies aimed at inhibiting KRAS oncogenic signaling by targeting either upstream activators or downstream effectors are also reviewed. Finally, we discuss the effect of targeting the mitogen‐activated protein kinase (MAPK) pathway, both based on the failure of MEK and ERK inhibitors in clinical trials, as well as on the recent identification of RAF1 as a potential target due to its MAPK‐independent activity. These new developments, taken together, are likely to open new avenues to effectively treat KRAS mutant LUAD.

Soft tissue tumors with RAF1 fusion had been emerging as a group of tumors with peculiar histology and immunoprofile. While a case series and rare case reports of RAF1 translocated sarcoma had been reported, to our knowledge a primary bone tumor with RAF1 translocation and fusion partner with MAP4 had not been described in the literature. The patient was a 60-year-old lady, with strong family history of breast cancer, who presented with pathological fracture of right humerus. X-ray revealed a 9.7 cm juxta-articular lesion of the proximal humerus, which was expansile and lytic with a non-sclerotic well defined border distally, radiologically suggestive of a giant cell tumor of bone. Excision was performed after initial biopsy. Histology showed a monomorphic low grade spindle cell lesion with prominent hyalinized stroma. Immunohistochemistry demonstrated diffuse CD34 staining, with focal staining for S100. Gene sequencing for histone 3 H3 genes was negative for hotspot mutation. Targeted RNA-seq sequencing revealed the presence of MAP4::RAF1 fusion, which was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH) break-apart probes involving both genes. The overall features were consistent with a primary bone sarcoma with RAF1 fusion. This report expanded the spectrum of RAF1 fusion sarcoma and was the first report documenting its primary occurrence in bone. [ABSTRACT FROM AUTHOR]

GNAQ, GNA11, and GNA14 are paralogues in the GNAQ family that are mutually exclusively mutated in a group of vascular neoplasms with thin-walled capillary-like vasoformative growth, which occur at multiple sites. The primary liver tumor in this category is termed hepatic small vessel neoplasm (HSVN), which is a rare infiltrative vascular neoplasm that can be misdiagnosed as hepatic angiosarcoma and which has been described as having likely very low malignant potential. In this study, we report on a novel primary malignant hepatic vascular neoplasm with infiltrative low grade regions morphologically resembling HSVN, as well as regions suggestive of high grade transformation. Next generation sequencing demonstrated a GNA11 p.Gln209Leu mutation (in high grade regions) and a RAF1 p.Met469Ile mutation (in low grade and high grade regions). On radiographic and gross examination, the tumor was unencapsulated, poorly circumscribed, and multifocal throughout the liver. Splenic metastases were identified and were histologically similar to high grade liver tumor regions, and shared the same RAF1 mutation. These findings argue that high grade morphology and/or additional oncogenic mutations in hepatic vascular neoplasms with GNAQ family mutations heighten concern for malignant potential and we advise next generation sequencing of HSVN for risk stratification.

Background: Lymphatic metastasis was an independent prognostic risk factor for hypopharyngeal carcinoma and was the main cause of treatment failure. The purpose of this study was to screen the differential genes and investigate the mechanism of lymphatic metastasis in hypopharyngeal carcinoma. Methods: Transcriptome sequencing was performed on primary tumors of patients, and differential genes were screened by bioinformatics analysis. The expression of differential genes was verified by qRT-PCR, western-blotting and immunohistochemical, and prognostic value was analyzed by Kaplan–Meier and log-rank test and Cox's test. Next, FADU and SCC15 cell lines were used to demonstrate the function of differential genes both in vitro by EdU, Flow cytometry, Wound Healing and Transwell assays and in vivo by a foot-pad xenograft mice model. Proteomic sequencing was performed to screen relevant targets. In addition, in vitro and in vivo experiments were conducted to verify the mechanism of lymphatic metastasis. Results: Results of transcriptome sequencing showed that RAF1 was a significantly differential gene in lymphatic metastasis and was an independent prognostic risk factor. In vitro experiments suggested that decreased expression of RAF1 could inhibit proliferation, migration and invasion of tumor cells and promote apoptosis. In vivo experiments indicated that RAF1 could promote tumor growth and lymphatic metastasis. Proteomic sequencing and subsequent experiments suggested that LAGE1 could promote development of tumor and lymphatic metastasis, and was regulated by RAF1. Conclusions: It suggests that RAF1 can promote lymphatic metastasis of hypopharyngeal carcinoma by regulating LAGE1, and provide a basis for the exploring of novel therapeutic target and ultimately provide new guidance for the establishment of intelligent diagnosis and precise treatment of hypopharyngeal carcinoma. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the second most common neurodegenerative disorder, and the mechanism of its occurrence is still not fully elucidated. Accumulating evidence has suggested that the gut acts as a potential origin of PD pathogenesis. Recent studies have identified that inflammatory bowel disease acts as a risk factor for Parkinson's disease, although the underlying mechanisms remain elusive. The aim of this study was to further explore the molecular mechanism between PD and Crohn's disease (CD). The gene expression profiles of PD (GSE6613) and CD (GSE119600) were downloaded from the Gene Expression Omnibus (GEO) database and were identified as the common differentially expressed genes (DEGs) between the two diseases. Next, analyses were performed, including functional enrichment analysis, a protein–protein interaction network, core genes identification, and clinical correlation analysis. As a result, 178 common DEGs (113 upregulated genes and 65 downregulated genes) were found between PD and CD. The functional analysis found that they were enriched in regulated exocytosis, immune response, and lipid binding. Twelve essential hub genes including BUB1B, BUB3, DLGAP5, AURKC, CBL, PCNA, RAF1, LYN, RPL39L, MRPL13, RSL24D1, and MRPS11 were identified from the PPI network by using cytoHubba. In addition, inflammatory and metabolic pathways were jointly involved in these two diseases. After verifying expression levels in an independent dataset (GSE99039), a correlation analysis with clinical features showed that LYN and RAF1 genes were associated with the severity of PD. In conclusion, our study revealed the common pathogenesis of PD and CD. These common pathways and hub genes may provide novel insights for mechanism research. [ABSTRACT FROM AUTHOR]

RAS proteins are binary switches, cycling between ON and OFF states during signal transduction. These switches are normally tightly controlled, but in RAS-related diseases, such as cancer, RASopathies, and many psychiatric disorders, mutations in the RAS genes or their regulators render RAS proteins persistently active. The structural basis of the switch and many of the pathways that RAS controls are well known, but the precise mechanisms by which RAS proteins function are less clear. All RAS biology occurs in membranes: a precise understanding of RAS' interaction with membranes is essential to understand RAS action and to intervene in RAS-driven diseases.

Mounting evidence suggests that circular RNAs play important roles in the development and progression of cancers. However, their function in glioblastomas (GBM) is still unclear. By circRNA array analysis, we found that circXPO1 (hsa_circ_102737) was significantly upregulated in GBM, and qPCR analysis verified that the circXPO1 expression level was increased in both GBM tissues and cell lines. Functional studies demonstrated that the knockdown of circXPO1 in GBM cell lines repressed cell proliferation and migration; conversely, the overexpression of circXPO1 promoted the malignancy of GBM cells. In line with these findings, circXPO1 inhibition effectively suppressed gliomagenesis in the in situ transplantation model of nude mice. Through bioinformatic analyses and dual-luciferase reporter assays, we showed that circXPO1 directly bound to miR-7-5p, which acted as a tumor suppressor through the negative regulation of RAF1. In conclusion, our studies suggest that the circXPO1/miR-7-5p/RAF1 axis promotes brain tumor formation and may be a potential therapeutic target for GBM treatment.

For decades, KRAS mutant lung adenocarcinomas (LUAD) have been refractory to therapeutic strategies based on personalized medicine owing to the complexity of designing inhibitors to selectively target KRAS and downstream targets with acceptable toxicities. The recent development of selective KRASG12C inhibitors represents a landmark after 40 years of intense research efforts since the identification of KRAS as a human oncogene. Here, we discuss the mechanisms responsible for the rapid development of resistance to these inhibitors, as well as potential strategies to overcome this limitation. Other therapeutic strategies aimed at inhibiting KRAS oncogenic signaling by targeting either upstream activators or downstream effectors are also reviewed. Finally, we discuss the effect of targeting the mitogen‐activated protein kinase (MAPK) pathway, both based on the failure of MEK and ERK inhibitors in clinical trials, as well as on the recent identification of RAF1 as a potential target due to its MAPK‐independent activity. These new developments, taken together, are likely to open new avenues to effectively treat KRAS mutant LUAD. [ABSTRACT FROM AUTHOR]

Background: Acinar cell carcinoma of the pancreas (ACC) is a rare malignancy, accounting for approximately 1-2% of all pancreatic cancers. Up to a quarter of ACCs are estimated to contain a significant component of another histology such as neuroendocrine or ductal adenocarcinoma. Prognosis and management remain uncertain for these mixed tumors. Case: A 71-year-old was initially diagnosed with biopsy-proven metastatic pancreatic ACC and treated with FOLFOX. Subsequent biopsy of a progressing metastatic site revealed ductal adenocarcinoma, and liquid biopsy showed a PRKAR1A-RAF1 fusion. The patient had a partial response to FOLFOX and died nine months after initial diagnosis. Discussion: This case highlights a metastatic presentation of mixed pancreatic acinar cell-ductal adenocarcinoma, its reasonable response to treatment with FOLFOX, and overall survival which was driven by the more aggressive ductal component. We also report the presence of a RAF1 rearrangement, which may present a possible therapeutic target in these rare tumors.

Purpose: The RAF1 expression affects prognosis in patients with hepatocellular carcinoma (HCC) treated with sorafenib. We examined the expression of sorafenib-targeted gene RAF1 to ascertain its relationship with gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid-enhanced magnetic resonance imaging (MRI) characteristics. We also explored the predictive potential of RAF1 expression markers in the treatment of HCC. Patients and methods: Sixty-five patients with HCC who underwent preoperative enhanced MRI scanning were included in this study. We analyzed the qualitative and quantitative characteristics of enhanced MRI findings in patients with HCC. Immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) analyses were used to detect the protein and mRNA expression levels of RAF1 in HCC. Correlation and logistic regression analyses were used to evaluate the relationship between these image features and the RAF1 gene expression levels in HCC. Results: The IHC analysis indicated a significant difference in tumor thrombus group (P = 0.037), RT-PCR results revealed a significant between-group difference for both tumor margins (P = 0.033) and capsule (P = 0.04). Binary logistic regression analysis results suggest that independent MRI predictors were regular tumor margins [P = 0.035, odds ratio (OR) = 3.145, 95% confidence interval (CI) 1.087–9.103] and thrombus (P = 0.046, OR = 4.421, 95% CI 1.024–19.08) with high RAF1expression; the tumor capsule was not an independent predictor. Conclusion: We found a correlation between MRI features and the RAF1 gene expression, Regular tumor margin and the presence of tumor thrombus are indicators of high RAF1 expression in HCC. Enhanced MRI may be useful for identifying patients with HCC eligible for targeted treatment. [ABSTRACT FROM AUTHOR]

Ameloblastoma is an odontogenic neoplasm characterized by slow intraosseous growth with progressive jaw resorption. Recent reports have revealed that ameloblastoma harbours an oncogenic BRAFV600E mutation with mitogen‐activated protein kinase (MAPK) pathway activation and described cases of ameloblastoma harbouring a BRAFV600E mutation in which patients were successfully treated with a BRAF inhibitor. Therefore, the MAPK pathway may be involved in the development of ameloblastoma; however, the precise mechanism by which it induces ameloblastoma is unclear. The expression of ADP‐ribosylation factor (ARF)‐like 4c (ARL4C), induced by a combination of the EGF–MAPK pathway and Wnt/β‐catenin signalling, has been shown to induce epithelial morphogenesis. It was also reported that the overexpression of ARL4C, due to alterations in the EGF/RAS–MAPK pathway and Wnt/β‐catenin signalling, promotes tumourigenesis. However, the roles of ARL4C in ameloblastoma are unknown. We investigated the involvement of ARL4C in the development of ameloblastoma. In immunohistochemical analyses of tissue specimens obtained from 38 ameloblastoma patients, ARL4C was hardly detected in non‐tumour regions but tumours frequently showed strong expression of ARL4C, along with the expression of both BRAFV600E and RAF1 (also known as C‐RAF). Loss‐of‐function experiments using inhibitors or siRNAs revealed that ARL4C elevation depended on the RAF1–MEK/ERK pathway in ameloblastoma cells. It was also shown that the RAF1–ARL4C and BRAFV600E–MEK/ERK pathways promoted cell proliferation independently. ARL4C‐depleted tumour cells (generated by knockdown or knockout) exhibited decreased proliferation and migration capabilities. Finally, when ameloblastoma cells were co‐cultured with mouse bone marrow cells and primary osteoblasts, ameloblastoma cells induced osteoclast formation. ARL4C elevation in ameloblastoma further promoted its formation capabilities through the increased RANKL expression of mouse bone marrow cells and/or primary osteoblasts. These results suggest that the RAF1–MEK/ERK–ARL4C axis, which may function in cooperation with the BRAFV600E–MEK/ERK pathway, promotes ameloblastoma development. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A subset of spindle cell tumours have been recently identified to harbor recurrent fusion genes, involving NTRK1/2/3, BRAF, RAF1, and RET. The precise classification of these fusion-positive tumours relies essentially on genomic profiling. Herein, we present our experience with two cases of spindle cell tumour which showed RAF1 rearrangement. Both tumours occurred in children with one in the left cheek (case 1) and the other one in the left buttock (case 2). Histologically, case 1 was a low-grade neoplasm characterized by uniform ovoid to short spindle cells showing "patternless" architecture with stromal hyalinization. Case 2 had an overtly malignant phenotype composed of long intersecting fascicles with increased cellularity and mitotic activity. By immunohistochemistry, tumour cells in case 1 showed co-expression of CD34 and S100 protein whereas in case 2 there was only focal staining of CD34 with no expression of S100 protein. Fluorescence in situ hybridization tests using NTRK1/2/3 (case 1 and case 2), ETV6, SS18, BRAF, ROS1, and ALK (case 2) break-apart probes were performed but yielded negative results. Subsequent next-generation sequencing (NGS) demonstrated PDZRN3-RAF1 fusion in case 1 and FMR1-RAF1 fusion in case 2, respectively, which were confirmed by FISH using RAF1 break-apart probe. This study further emphasizes the importance of molecular diagnostics in fusion-positive spindle cell tumours. In addition, we expand the genetic spectrum of RAF1-rearranged spindle cell tumour by describing a novel FMR1-RAF1 fusion gene. [ABSTRACT FROM AUTHOR]

Bashan Zhang, Fei Li, Zinian Zhu, Aijiao Ding, Jintong Luo Department of Clinical Laboratory, Affiliated Dongguan People’s Hospital, Southern Medical University of Dongguan, Dongguan City, Guangdong Province, People’s Republic of ChinaCorrespondence: Bashan ZhangDepartment of Clinical Laboratory, Affiliated Dongguan People’s Hospital, Southern Medical University of Dongguan, Wanjiang District, Dongguan City, Guangdong Province 523059, People’s Republic of ChinaTel +86 769 28636657Email zrz7hit@163.comBackground: Hepatocellular carcinoma (HCC) is a common lethal malignant tumor worldwide. Circular RNAs (circRNAs) have been reported to affect the development of human cancers, including HCC. In this project, we aim to clarify the functional effect of circular CDR1as (circ_CDR1as) on HCC progression.Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot is implemented to detect the expression of circ_CDR1as, microRNA (miR)-1287 and Raf-1 proto-oncogene, serine/threonine kinase (Raf1). Cell proliferation is assessed via colony formation and 3-(4, 5)-dimethylthiazole-2-y1)-2, 5-biphenyl tetrazolium bromide (MTT) assays. Cell migration and invasion are measured by Transwell assay. The target relationship between miR-1287 and circ_CDR1as or Raf1 is validated through dual-luciferase reporter assay. The levels of epithelia–mesenchymal transition (EMT) markers and the MEK/ERK signal pathway-related proteins are examined by Western blot. Model in nude mice is constructed to determine the role of circ_CDR1as in vivo.Results: Expression of circ_CDR1as and Raf1 is elevated, while miR-1287 expression is decreased in HCC. Depletion of circ_CDR1as or Raf1 could inhibit proliferation and metastasis of HCC cells. Besides, circ_CDR1as regulates Raf1 expression by targeting miR-1287. MiR-1287 upregulation or Raf1 depletion could partially counterbalance circ_CDR1as depletion-mediated inhibitory effects on HCC cell behaviors. Moreover, circ_CDR1as depletion represses HCC progression through inactivating MEK/ERK pathway. In addition, circ_CDR1as depletion suppresses tumor growth in vivo via regulating miR-1287/Raf1 pathway.Conclusion: Circ_CDR1as depletion inhibits HCC cell proliferation and metastasis by miR-1287/Raf1 and MEK/ERK pathways, highlighting a promising molecular target for HCC treatment.Keywords: HCC, circ_CDR1as, Raf1, miR-1287, proliferation, metastasis, MEK/ERK pathway

Abstract Background Papillary thyroid cancer (PTC) is the most common malignancy of all thyroid cancers. LncRNA LINC00460 has been proved to play roles in the oncogenesis and progression of various tumors, including papillary thyroid cancer. However, the potential molecular mechanism of LINC00460 in PTC is poorly investigated. Results LINC00460 was upregulated in PTC tissues and cells. Raf1 was upregulated in PTC tissues, but miR-485-5p was down-regulated. High LINC00460 expression was associated with poor prognosis. LINC00460 knockdown suppressed proliferation, migration, invation and EMT of PTC cells. Bioinformatics prediction revealed that LINC00460 had binding sites with miR-485-5p, which was validated by luciferase reporter assay. In addition, miR-485-5p was confirmed to directly target Raf1 3′-UTR. Moreover, LINC00460 promoted PTC progression by sponging miR-485-5p to elevate the expression of Raf1. Knockdown of LINC00460 restrained tumor growth in vivo. Conclusion LINC00460 induced proliferation, migration, invation and EMT of PTC cells by regulating the LINC00460/miR-485-5p/Raf1 axis, which indicated that LINC00460 may be a potential biomarker and therapeutic target for PTC.

Abstract Background In past decades, circular RNAs (circRNAs) have achieved increasing attention because of its regulatory role in different kinds of cancers. However, how circAGFG1 regulates cervical cancer (CC) is still largely undiscovered. This study aims to evaluate the role of a novel circRNAs and related molecular mechanism in CC cells. Methods High or low level of circAGFG1 was detected in CC cells or normal cell line with qRT-PCR. The proliferative and migratory abilities of CC cells were assessed with loss-of function assays. The downstream miRNA and mRNA of circAGFG1 were searched out and proved by using bioinformatics analysis and mechanism experiments. Recue assays were designed to confirm the role of circAGFG1/miR-370-3p/RAF1 axis in CC cell activities. Results The levels of circAGFG1 was abundant in CC cells in comparison with normal cervical cell End1/E6E7. The inhibitory effect of decreased circAGFG1 level on the proliferative and migratory abilities of CC cells was assessed. CircAGFG1 and miR-370-3p were localized in the cytoplasm and they can interact with each other. Moreover, miR-370-3p was downregulated in CC cells. We also determined the negative effect of miR-370-3p on RAF1. CircAGFG1 could promote RAF1 expression by absorbing miR-370-3p, thereby activating RAF/MEK/ERK pathway. circAGFG1 promoted proliferation and migration of CC cells via enhancing the activity of RAF/MEK/ERK pathway by sponging miR-370-3p and further regulating RAF1. Conclusion The results of this study provided new evidence that circAGFG1 acted as a vital regulator in cervical cancer proliferation and migration, giving great promise to apply it as a potential biomarker for diagnosis and therapy in CC treatment.

Approved BRAF inhibitors have shown limited clinical benefit due to recurrent disease progression. In a recent Cancer Discovery paper, Yaeger et al. show that a next-generation BRAF inhibitor, PF-07799933, has widespread therapeutic activity in experimental models and patients who were refractory to treatment with approved BRAF inhibitors., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The diagnosis of cutaneous and subcutaneous spindle cell neoplasms in children is often challenging and has potential therapeutic and prognostic implications. Although correctly diagnosing dermatofibrosarcoma protuberans and infantile fibrosarcoma is paramount, pathologists should not ignore a number of diagnostic pitfalls linked to mostly rare tumors with completely different clinical outcomes. In the last decade, a spectrum of novel entities has been described; information from molecular biology has helped to shape this new landscape for spindle cell tumors. Here, we review the most noteworthy neoplasms in this spectrum, with a focus on their histological similarities: fibroblastic connective tissue nevus, medallion-like dermal dendrocyte hamartoma, or plaque-like CD34-positive dermal fibroma, which share features with fibrous hamartoma of infancy; lipofibromatosis and lipofibromatosis-like neural tumor; and plexiform myofibroblastoma, a recently described neoplasm that should be distinguished from plexiform fibrohistiocytic tumor. These tumors also have genetic similarities, particularly gene rearrangements involving NTRK3 or NTRK1. These genetic features are not only essential for the differential diagnosis of infantile fibrosarcoma but are also of diagnostic value for lipofibromatosis-like neural tumors. The more recently described RET, RAF1, and BRAF gene fusions are also discussed. [ABSTRACT FROM AUTHOR]

Both microRNA-7a (miR-7a) and LIM-homeodomain transcription factor ISL1 are important factors regulating insulin transcription and secretion, but the functional relationship and the interacting mechanisms between miR-7a and ISL1 in pancreatic islet β-cells remain unknown. The aims of this study were thus to identify the potential interactions and signaling communication between miR-7a and ISL1 in regulating insulin transcription and secretion in the cultured NIT-1 cells. The results show that miR-7a inhibitor upregulates Isl-1 and insulin gene expressions, and the insulin secretion. Whereas miR-7a mimics inhibit ISL1 and insulin gene expressions, and decreases the insulin secretion. Furthermore, we identified the target gene of miR-7a using dual-luciferase reporter assay, and the results demonstrate that Raf1 and Mapkap1 is a direct target gene of miR-7a, modeling RAF1/MEK/ERK1/2 and mTORC2/AKT signaling pathway to regulate Isl1 expression, and thus influencing insulin expression and secretion. Our results indicate that therapeutic inhibition of miR-7a function could be of relevance for preserving the function of pancreatic β-cells during the course of diabetes development, implicating miR-7, ISL1, and/or the connecting molecules may act as novel targets for pharmacological or gene therapy in diabetes and related metabolic disease, although much detailed studies are required in the further study. [ABSTRACT FROM AUTHOR]

Background: Liver cancer is a major public health concern, but the mechanistic actions of biomarkers contributing to liver cancer remain to be determined. In this study, we aimed to investigate the regulatory cascade of microRNA‐424‐5p (miR‐424‐5p), X‐inactive‐specific transcript (XIST) and O‐GlcNAc transferase (OGT) in liver cancer. Methods: Differentially expressed miRNAs and target genes related to liver cancer were predicted by bioinformatics analyses, and their expression was determined in liver tissues of patients with liver cancer and liver cancer cells. The RNA immunoprecipitation (RIP), RNA pull‐down and dual luciferase reporter assay were used to examine the binding affinity among XIST and miR‐424‐5p and OGT. Then, gain‐ and loss‐of‐function assays were conducted to evaluate the effects of the XIST/miR‐424‐5p/OGT axis on malignant phenotypes. A nude mouse model of liver cancer was further established for in vivo substantiation. Results: XIST and OGT were up‐regulated in liver cancer tissues and cells, responsible for poor prognosis in patients with liver cancer, while miR‐424‐5p was down‐regulated. XIST competitively bound to miR‐424‐5p to increase OGT expression. XIST silencing inhibited malignant phenotypes of liver cancer cells, while miR‐424‐5p down‐regulation negated its effect. miR‐424‐5p suppressed RAF1 glycosylation by negatively regulating OGT expression and promoted its ubiquitination/degradation. Furthermore, XIST knockdown inhibited tumour growth and metastasis in nude mice, while ectopic OGT reversed its effect. Conclusion: These results reveal a novel mechanism by which the interaction of XIST/miR‐424‐5p/OGT participates in the malignancy and metastasis of liver cancer. [ABSTRACT FROM AUTHOR]

Background and Purpose: Bronchopulmonary dysplasia (BPD) is the most prevalent chronic paediatric lung disease and is linked to the development of chronic obstructive pulmonary disease. MicroRNA-based regulation of type II alveolar epithelial cell (T2AEC) proliferation and apoptosis is an important factor in the pathogenesis of BPD and warrants further investigation.Experimental Approach: Two murine models of hyperoxic lung injury (with or without miR-342-5p or Sprouty-related, EVH1 domain-containing protein 3 [Spred3] modulation) were employed: a hyperoxia-induced acute lung injury model (100% O2 on postnatal days 1-7) and the BPD model (100% O2 on postnatal days 1-4, followed by room air for 10 days). Tracheal aspirate pellets from healthy control and moderate/severe BPD neonates were randomly selected for clinical miR-342-5p analysis.Key Results: Hyperoxia decreased miR-342-5p levels in primary T2AECs, MLE12 cells and neonatal mouse lungs. Transgenic miR-342 overexpression in neonatal mice ameliorated survival rates and improved the BPD phenotype and BPD-associated pulmonary arterial hypertension (PAH). T2AEC-specific miR-342 transgenic overexpression, as well as miR-342-5p mimic therapy, also ameliorated the BPD phenotype and associated PAH. miR-342-5p targets the 3'UTR of the Raf1 regulator Spred3, inhibiting Spred3 expression. Treatment with recombinant Spred3 exacerbated the BPD phenotype and associated PAH. Notably, miR-342-5p inhibition under room air conditions did not mimic the BPD phenotype. Moderate/severe BPD tracheal aspirate pellets exhibited decreased miR-342-5p levels relative to healthy control pellets.Conclusion and Implications: These findings suggest that miR-342-5p mimic therapy may show promise in the treatment or prevention of BPD. [ABSTRACT FROM AUTHOR]

Abstract Background p21-activated kinase 1 (PAK1) plays a fundamental role in promoting the development and progression of several cancers and is a potential therapeutic target. However, the biological function and underlying mechanism of PAK1 in esophageal squamous cell carcinoma (ESCC) remain unclear. Methods The expression of PAK1 was detected in both ESCC cell lines and clinical samples. Cell growth was measured by MTT, focus formation and soft agar assays. Cell migration and invasion were detected by wound healing and transwell assays. Animal models of subcutaneous tumourigenicity and tail vein metastasis were performed to determine the inhibitory effect of pharmacological inhibitor IPA-3 on tumor growth and metastasis of ESCC cells. Results We found that PAK1 was frequently overexpressed in ESCC. Ectopic expression of PAK1 promoted cellular growth, colony formation and anchorage-independent growth. Overexpressing PAK1 also enhanced migration, invasion and the expression of MMP-2 and MMP-9 in ESCC cells. In contrast, silencing PAK1 by lentiviral knockdown or a specific inhibitor IPA-3 resulted in a contrary effect. Subsequent investigations revealed that Raf1/MEK1/ERK signaling pathway was involved in PAK1-mediated effect. Enhanced expression of Raf1 attenuated the inhibitory functions of PAK1 shRNA. Whereas blocking of Raf1 by shRNA or specific inhibition of MEK1 by U0126 antagonized the oncogenetic effect of PAK1 on ESCC cells. More importantly, Pharmacological inhibition of PAK1 by IPA-3 significantly suppressed tumor growth and lung metastasis of ESCC cells in vivo. Conclusions These data support that PAK1 is an ideal target for the development of potential therapeutic drugs for ESCC patients even with metastasis.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, and the mechanism of its occurrence is still not fully elucidated. Accumulating evidence has suggested that the gut acts as a potential origin of PD pathogenesis. Recent studies have identified that inflammatory bowel disease acts as a risk factor for Parkinson’s disease, although the underlying mechanisms remain elusive. The aim of this study was to further explore the molecular mechanism between PD and Crohn’s disease (CD). The gene expression profiles of PD (GSE6613) and CD (GSE119600) were downloaded from the Gene Expression Omnibus (GEO) database and were identified as the common differentially expressed genes (DEGs) between the two diseases. Next, analyses were performed, including functional enrichment analysis, a protein–protein interaction network, core genes identification, and clinical correlation analysis. As a result, 178 common DEGs (113 upregulated genes and 65 downregulated genes) were found between PD and CD. The functional analysis found that they were enriched in regulated exocytosis, immune response, and lipid binding. Twelve essential hub genes including BUB1B, BUB3, DLGAP5, AURKC, CBL, PCNA, RAF1, LYN, RPL39L, MRPL13, RSL24D1, and MRPS11 were identified from the PPI network by using cytoHubba. In addition, inflammatory and metabolic pathways were jointly involved in these two diseases. After verifying expression levels in an independent dataset (GSE99039), a correlation analysis with clinical features showed that LYN and RAF1 genes were associated with the severity of PD. In conclusion, our study revealed the common pathogenesis of PD and CD. These common pathways and hub genes may provide novel insights for mechanism research.

Prolactin production is controlled by a complex and temporally dynamic network of factors. Despite this tightly coordinated system, pathological hyperprolactinemia is a common endocrine disorder that is often not understood, thereby highlighting the need to expand our molecular understanding of lactotroph cell regulation. MicroRNA-7 (miR-7) is the most highly expressed miRNA family in the pituitary gland and the loss of the miR-7 family member, miR-7a2, is sufficient to reduce prolactin gene expression in mice. Here, we used conditional loss-of-function and gain-of-function mouse models to characterize the function of miR-7a2 in lactotroph cells. We found that pituitary miR-7a2 expression undergoes developmental and sex hormone–dependent regulation. Unexpectedly, the loss of mir-7a2 induces a premature increase in prolactin expression and lactotroph abundance during embryonic development, followed by a gradual loss of prolactin into adulthood. On the other hand, lactotroph development is delayed in mice overexpressing miR-7a2. This regulation of lactotroph function by miR-7a2 involves complementary mechanisms in multiple cell populations. In mouse pituitary and rat prolactinoma cells, miR-7a2 represses its target Raf1, which promotes prolactin gene expression. These findings shed light on the complex regulation of prolactin production and may have implications for the physiological and pathological mechanisms underlying hyperprolactinemia. [ABSTRACT FROM AUTHOR]

HIV cure is thwarted by the presence of quiescent yet replication competent HIV-1 (HIV). Antiretroviral therapy (ART) is unable to eradicate reservoirs, and upon cessation of ART, HIV will rebound. This review encompasses the curative strategies of HIV in the context of NF-κB sub-pathways that are currently exploited and demonstrate promise in the disruption of latent HIV. Canonical NF-κB signaling has long been established to drive HIV proviral expression while noncanonical NF-κB signaling, a novel and perhaps more desirable mechanism of latency reversal due to its unique characteristics, has recently been shown to also promote HIV expression from latency. Furthermore, we discuss the previously unrecognized upstream signaling of NF-κB as a new avenue for exploration of a functional cure of HIV.

Since the discovery of the ETV6-NTRK3 gene fusion in infantile fibrosarcoma two decades ago, it has become an important diagnostic marker because it is found in the majority of cases. However, the development of new molecular tests, including next generation sequencing, has uncovered additional gene fusions and other oncogenic mutations in tumors with the clinical and morphologic features of infantile fibrosarcoma. We present a case of infantile fibrosarcoma harboring a novel BMPR1A-RAF1 fusion and having a favorable outcome 6 years after surgery. This new structural alteration adds to the list of genetic aberrations in infantile fibrosarcoma and provides another example of the challenge of reconciling classic morphology with novel molecular genetics.

The RAF kinase inhibitor protein, RKIP, is a dual inhibitor of the RAF1 kinase and the G protein-coupled receptor kinase 2, GRK2. By inhibition of the RAF1-MAPK (mitogen-activated protein kinase) pathway, RKIP acts as a beneficial tumour suppressor. By inhibition of GRK2, RKIP counteracts GRK2-mediated desensitisation of G protein-coupled receptor (GPCR) signalling. GRK2 inhibition is considered to be cardioprotective under conditions of exaggerated GRK2 activity such as heart failure. However, cardioprotective GRK2 inhibition and pro-survival RAF1-MAPK pathway inhibition counteract each other, because inhibition of the pro-survival RAF1-MAPK cascade is detrimental for the heart. Therefore, the question arises, what is the net effect of these apparently divergent functions of RKIP in vivo? The available data show that, on one hand, GRK2 inhibition promotes cardioprotective signalling in isolated cardiomyocytes. On the other hand, inhibition of the pro-survival RAF1-MAPK pathway by RKIP deteriorates cardiomyocyte viability. In agreement with cardiotoxic effects, endogenous RKIP promotes cardiac fibrosis under conditions of cardiac stress, and transgenic RKIP induces heart dysfunction. Supported by next-generation sequencing (NGS) data of the RKIP-induced cardiac transcriptome, this review provides an overview of different RKIP functions and explains how beneficial GRK2 inhibition can go awry by RAF1-MAPK pathway inhibition. Based on RKIP studies, requirements for the development of a cardioprotective GRK2 inhibitor are deduced.

A small subset of cutaneous melanomas harbor oncogenic gene fusions, which could potentially serve as therapeutic targets for patients with advanced disease as novel therapies are developed. Fusions involving RAF1 are exceedingly rare in melanocytic neoplasms, occurring in less than 1% of melanomas, and usually arise in tumors that are wild type for BRAF, NRAS, and NF1. We describe herein a case of acral melanoma with two satellite metastases and sentinel lymph node involvement. The melanoma had a concomitant KIT variant and LRRFIP2‐RAF1 fusion. This constellation of molecular findings has not been reported previously in melanoma. We review the existing literature on melanocytic neoplasms with RAF1 fusions and discuss the potential clinical implications of this genetic event. [ABSTRACT FROM AUTHOR]

Abstract Background RAF family activating fusions have been described as a potentially targetable molecular finding in a subset of soft tissue sarcomas. To further expand upon the landscape of this genetic feature, we describe a novel MTAP-RAF1 activating fusion identified in a S100 positive soft tissue sarcoma. Case presentation A 51 year old man underwent excision of a soft tissue mass in his foot. Pathology revealed a spindle cell neoplasm with S100 positivity, ultimately classified as a soft tissue sarcoma, not otherwise specified. Comprehensive molecular profiling was performed to help establish the diagnosis and revealed a novel MTAP-RAF1 fusion that includes the tyrosine kinase domain of RAF1. Conclusions Our report adds to the spectrum of fusion-driven RAF activation observed in soft tissue sarcomas and lends additional evidence that RAF activation plays an important role in some soft tissue sarcomas. Identification of novel fusions involving the MAPK/ERK pathway in sarcomas may provide new avenues for precision medicine strategies involving targeted kinase inhibitors.