FTO-mediated SMAD2 m6A modification protects cartilage against Osteoarthritis.

N6-methyladenosine (m6A) modification is one of the most prevalent forms of epigenetic modification and plays an important role in the development of degenerative diseases such as osteoarthritis (OA). However, the evidence concerning the role of m6A modification in OA is insufficient. Here, m6A modification was increased in human OA cartilage and degenerated chondrocytes. Among all of the m6A enzymes, the expression of the demethylase fat mass and obesity-associated protein (FTO) decreased dramatically. Conditional knockout of FTO in chondrocytes accelerates OA progression. FTO transcription is regulated by runt-related transcription factor-1 (RUNX1). Reduced FTO elevates m6A modification at the adenosine N6 position in SMAD family member 2 (SMAD2) mRNA, whose stability is subsequently modulated by the recruited m6A reader protein YTH N6-methyladenosine RNA binding protein F2 (YTHDF2). Collectively, these findings reveal the function and mechanism of the m6A family member FTO in OA progression. Therefore, reducing m6A modification to increase SMAD2 stability by activating FTO might be a potential therapeutic strategy for OA treatment.

Loss of DDRGK1 impairs IRE1α UFMylation in spondyloepiphyseal dysplasia.

Spondyloepiphyseal dysplasia (SEMD) is a rare disease in which cartilage growth is disrupted, and the DDRGK1 mutation is one of the causative genes. In our study, we established Ddrgk1fl/fl, Col2a1-ERT Cre mice, which showed a thickened hypertrophic zone (HZ) in the growth plate, simulating the previous reported SEMD pathology in vivo. Instead of the classical modulation mechanism towards SOX9, our further mechanism study found that DDRGK1 stabilizes the stress sensor endoplasmic reticulum-to-nucleus signaling 1 (IRE1α) to maintain endoplasmic reticulum (ER) homoeostasis. The loss of DDRGK1 decreased the UFMylation and subsequently led to increased ubiquitylation-mediated IRE1α degradation, causing ER dysfunction and activating the PERK/CHOP/Caspase3 apoptosis pathway. Further DDRGK1 K268R-mutant mice revealed the importance of K268 UFMylation site in IRE1α degradation and subsequent ER dysfunction. In conclusion, DDRGK1 stabilizes IRE1α to ameliorate ER stress and following apoptosis in chondrocytes, which finally promote the normal chondrogenesis.

The cryo-EM structure of the human ERAD retrotranslocation complex.

Endoplasmic reticulum-associated degradation (ERAD) maintains protein homeostasis by retrieving misfolded proteins from the endoplasmic reticulum (ER) lumen into the cytosol for degradation. The retrotranslocation of misfolded proteins across the ER membrane is an energy-consuming process, with the detailed transportation mechanism still needing clarification. We determined the cryo-EM structures of the hetero-decameric complex formed by the Derlin-1 tetramer and the p97 hexamer. It showed an intriguing asymmetric complex and a putative coordinated squeezing movement in Derlin-1 and p97 parts. With the conformational changes of p97 induced by its ATP hydrolysis activities, the Derlin-1 channel could be torn into a "U" shape with a large opening to the lipidic environment, thereby forming an entry for the substrates in the ER membrane. The EM analysis showed that p97 formed a functional protein complex with Derlin-1, revealing the coupling mechanism between the ERAD retrotranslocation and the ATP hydrolysis activities.

Aminooxyacetic acid hemihydrochloride leads to decreased intracellular ATP levels and altered cell cycle of prostate cancer cells by suppressing energy metabolism.

The second most common cancer among men is prostate cancer, which is also the fifth leading reason for male cancer deaths worldwide. Bone metastases are the main factor affecting the prognosis of prostate cancer. Consequently, antitumor and anti-prostate cancer-induced bone destruction medicines are urgently needed. We previously discovered that aminooxyacetic acid hemihydrochloride (AOAA) suppressed bone resorption and osteoclast growth by decreasing adenosine triphosphate (ATP) production and limiting oxidative phosphorylation (OXPHOS). Here, we evaluated the impacts of AOAA on prostate cancer RM-1 cells in vitro. It's found that AOAA significantly inhibited cell proliferation, migration, and invasiveness, decreased ATP levels, increased ROS, halted the cell cycle phase, and triggered apoptosis. AOAA also decreased mitochondrial membrane potential and the ability to uptake glucose, suggesting that the antitumor effects of AOAA were expressed through the inhibition of OXPHOS and glycolysis. Furthermore, we assessed the effects of AOAA in vivo using a prostate cancer-induced bone osteolysis mice model. AOAA also delayed tumor growth and bone destruction in vivo. On the whole, our findings imply that AOAA may potentially have therapeutic effects on prostate cancer and prostate cancer-induced osteolysis.

Dlk2 interacts with Syap1 to activate Akt signaling pathway during osteoclast formation.

Excessive osteoclast formation and bone resorption are related to osteolytic diseases. Delta drosophila homolog-like 2 (Dlk2), a member of the epidermal growth factor (EGF)-like superfamily, reportedly regulates adipocyte differentiation, but its roles in bone homeostasis are unclear. In this study, we demonstrated that Dlk2 deletion in osteoclasts significantly inhibited osteoclast formation in vitro and contributed to a high-bone-mass phenotype in vivo. Importantly, Dlk2 was shown to interact with synapse-associated protein 1 (Syap1), which regulates Akt phosphorylation at Ser473. Dlk2 deletion inhibited Syap1-mediated activation of the AktSer473, ERK1/2 and p38 signaling cascades. Additionally, Dlk2 deficiency exhibits increased bone mass in ovariectomized mice. Our results reveal the important roles of the Dlk2-Syap1 signaling pathway in osteoclast differentiation and osteoclast-related bone disorders.

Cynarin alleviates intervertebral disc degeneration via protecting nucleus pulposus cells from ferroptosis.

Intervertebral disc degeneration (IVDD) leads to a series of degenerative spine diseases. Clinical treatment of IVDD is mainly surgery, lacking effective drugs to alleviate intervertebral disc degeneration. In this study, we analysed the mRNA sequencing dataset of human degenerative intervertebral disc tissues and revealed the participation of ferroptosis in IVDD. Furthermore, we confirmed that TNF-α, an important cytokine in IVDD, induces ferroptosis in nucleus pulposus cells. Subsequently, a ferroptosis inhibitors screening strategy using multiple ferroptosis indicators was developed. Through the screen of various natural compounds, cynarin, a natural product enriched in Artichoke, was discovered to inhibit ferroptosis of nucleus pulposus cells. Cynarin can dose-dependently inhibit the catabolism of nucleus pulposus cells, increase the expression of key ferroptosis-inhibiting genes (GPX4 and NRF2), inhibit the increment of cellular Fe2+, lipid peroxides, and reactive oxygen species. It can also prevent mitochondria shrinkage, reduce mitochondria cristae density in ferroptosis, and prevent IVDD in the rat model. In conclusion, cynarin is a potential candidate for the drug development for IVDD.

DDRGK1 Enhances Osteosarcoma Chemoresistance via Inhibiting KEAP1-Mediated NRF2 Ubiquitination.

Chemoresistance is the main obstacle in osteosarcoma (OS) treatment; however, the underlying mechanism remains unclear. In this study, it is discovered that DDRGK domain-containing protein 1 (DDRGK1) plays a fundamental role in chemoresistance induced in OS. Bioinformatic and tissue analyses indicate that higher expression of DDRGK1 correlates with advanced tumor stage and poor clinical prognosis of OS. Quantitative proteomic analyses suggest that DDRGK1 plays a critical role in mitochondrial oxidative phosphorylation. DDRGK1 knockout trigger the accumulation of reactive oxygen species (ROS) and attenuate the stability of nuclear factor erythroid-2-related factor 2 (NRF2), a major antioxidant response element. Furthermore, DDRGK1 inhibits ubiquitin-proteasome-mediated degradation of NRF2 via competitive binding to the Kelch-like ECH-associated protein 1 (KEAP1) protein, which recruits NRF2 to CULLIN(CUL3). DDRGK1 knockout attenuates NRF2 stability, contributing to ROS accumulation, which promotes apoptosis and enhanced chemosensitivity to doxorubicin (DOX) and etoposide in cancer cells. Indeed, DDRGK1 knockout significantly enhances osteosarcoma chemosensitivity to DOX in vivo. The combination of DDRGK1 knockdown and DOX treatment provides a promising new avenue for the effective treatment of OS.

Ceritinib (LDK378) prevents bone loss via suppressing Akt and NF-κB-induced osteoclast formation.

Background: Ceritinib is used for the treatment of patients with anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC), who are at the risk of developing bone metastasis. During bone metastasis, tumor cells release factors that induce osteoclast formation, resulting in osteolysis. However, the effect of ceritinib on osteoclast formation remains unclear. Methods: Osteoclastogenesis was induced to assess the effect of ceritinib on osteoclast formation and osteoclast-specific gene expression. Western blotting was used to examine the molecular mechanisms underlying the effect of ceritinib on osteoclast differentiation. An in vivo ovariectomized mouse model was established to validate the effect of ceritinib in suppressing osteoclast formation and preventing bone loss. Results: The differentiation of osteoclasts and the expression of osteoclast-specific genes were inhibited upon ceritinib stimulation. Ceritinib suppressed Akt and p65 phosphorylation during the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. The administration of ceritinib to ovariectomized mice ameliorated trabecular bone loss by inhibiting osteoclast formation. Conclusions: Ceritinib is beneficial in preventing bone loss by suppressing osteoclastic Akt and nuclear factor κB (NF-κB) signaling.

Specific PFKFB3 Inhibitor Memorably Ameliorates Intervertebral Disc Degeneration via Inhibiting NF-κB and MAPK Signaling Pathway and Reprogramming of Energy Metabolism of Nucleus Pulposus Cells.

Intervertebral disc (IVD) degeneration (IVDD) is a characteristic of the dominating pathological processes of nucleus pulposus (NP) cell senescence, abnormal synthesis and irregular distribution of extracellular matrix (ECM), and tumor necrosis factor-α (TNF-α) induced inflammation. Nowadays, IVD acid environment variation which accelerates the pathological processes mentioned above arouses researchers' attention. KAN0438757 (KAN) is an effective inhibitor of selective metabolic kinase phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) that has both energy metabolism reprogramming and anti-inflammatory effects. Therefore, a potential therapeutic benefit of KAN lies in its ability to inhibit the development of IVDD. This study examined in vitro KAN toxicity in NP primary cells (NPPs). Moreover, KAN influenced tumor necrosis factor-α (TNF-α) induced ECM anabolism and catabolism; the inflammatory signaling pathway activation and the energy metabolism phenotype were also examined in NPPs. Furthermore, KAN's therapeutic effect was investigated in vivo using the rat tail disc puncture model. Phenotypically speaking, the KAN treatment partially rescued the ECM degradation and glycolysis energy metabolism phenotypes of NPPs induced by TNF-α. In terms of mechanism, KAN inhibited the activation of MAPK and NF-κB inflammatory signaling pathways induced by TNF-α and reprogramed the energy metabolism. For the therapeutic aspect, the rat tail disc puncture model demonstrated that KAN has a significant ameliorated effect on the progression of IVDD. To sum up, our research successfully authenticated the potential therapeutic effect of KAN on IVDD and declaimed its mechanisms of both novel energy metabolism reprogramming and conventional anti-inflammation effect.

MR-based synthetic CT image for intensity-modulated proton treatment planning of nasopharyngeal carcinoma patients.

PURPOSE: To develop an advanced deep convolutional neural network (DCNN) architecture to generate synthetic CT (SCT) images from MR images for intensity-modulated proton therapy (IMPT) treatment planning of nasopharyngeal cancer (NPC) patients. METHODS: T1-weighted MR images and paired CT (PCT) images were obtained from 206 NPC patients. For each patient, deformable image registration was performed between MR and PCT images to create an MR-CT image pair. Thirty pairs were randomly chosen as the independent test set and the remaining 176 pairs (14 for validation and 162 for training) were used to build two conditional generative adversarial networks (GANs): 1) GAN3D: using a 3D U-net enhanced with residual connections and attentional mechanism as the generator and 2) GAN2D: using a 2D U-net as the generator. For each test patient, SCT images were generated using the generators with the MR images as input and were compared with respect to the corresponding PCT image. A clinical IMPT plan was created and optimized on the PCT image. The dose was recalculated on the SCT images and compared with the one calculated on the PCT image. RESULTS: The mean absolute errors (MAEs) between the PCT and SCT, within the body, were (64.89 ± 5.31) HU and (64.31 ± 4.61) HU for the GAN2D and GAN3D. Within the high-density bone (HU > 600), the GAN3D achieved a smaller MAE compared with the GAN2D (p < 0.001). Within the body, the absolute point dose deviation was reduced from (0.58 ± 1.61) Gy for the GAN2D to (0.47 ± 0.94) Gy for the GAN3D. The (3 mm/3%) gamma passing rates were above 97.32% for all SCT images. CONCLUSIONS: The SCT images generated using GANs achieved clinical acceptable dosimetric accuracy for IMPT of NPC patients. Using advanced DCNN architecture design, such as residual connections and attention mechanism, SCT image quality was further improved and resulted in a small dosimetric improvement.

Current applications of adipose-derived mesenchymal stem cells in bone repair and regeneration: A review of cell experiments, animal models, and clinical trials.

In the field of orthopaedics, bone defects caused by severe trauma, infection, tumor resection, and skeletal abnormalities are very common. However, due to the lengthy and painful process of related surgery, people intend to shorten the recovery period and reduce the risk of rejection; as a result, more attention is being paid to bone regeneration with mesenchymal stromal cells, one of which is the adipose-derived mesenchymal stem cells (ASCs) from adipose tissue. After continuous subculture and cryopreservation, ASCs still have the potential for multidirectional differentiation. They can be implanted in the human body to promote bone repair after induction in vitro, solve the problems of scarce sources and large damage, and are expected to be used in the treatment of bone defects and non-union fractures. However, the diversity of its differentiation lineage and the lack of bone formation potential limit its current applications in bone disease. Here, we concluded the current applications of ASCs in bone repair, especially with the combination and use of physical and biological methods. ASCs alone have been proved to contribute to the repair of bone damage in vivo and in vitro. Attaching to bone scaffolds or adding bioactive molecules can enhance the formation of the bone matrix. Moreover, we further evaluated the efficiency of ASC-committed differentiation in the bone in conditions of cell experiments, animal models, and clinical trials. The results show that ASCs in combination with synthetic bone grafts and biomaterials may affect the regeneration, augmentation, and vascularization of bone defects on bone healing. The specific conclusion of different materials applied with ASCs may vary. It has been confirmed to benefit osteogenesis by regulating osteogenic signaling pathways and gene transduction. Exosomes secreted by ASCs also play an important role in osteogenesis. This review will illustrate the understanding of scientists and clinicians of the enormous promise of ASCs' current applications and future development in bone repair and regeneration, and provide an incentive for superior employment of such strategies.

Ano5 modulates calcium signaling during bone homeostasis in gnathodiaphyseal dysplasia.

ANO5 encodes transmembrane protein 16E (TMEM16E), an intracellular calcium-activated chloride channel in the endoplasmic reticulum. Mutations in ANO5 are associated with gnathodiaphyseal dysplasia (GDD), a skeletal disorder causing the jaw deformity and long bone fractures. However, the coordinated mechanism by which ANO5 mediates bone homeostasis in GDD remains poorly defined. Here, we show that ablation of Ano5 reduced intracellular calcium transients, leading to defects in osteogenesis and osteoclastogenesis and thus bone dysplasia. We found a causative de novo ANO5 frameshift insertion mutation (p.L370_A371insDYWRLNSTCL) in a GDD family with osteopenia, accompanied by a decrease in TMEM16E expression and impaired RANKL-induced intracellular calcium ([Ca2+]i) oscillations in osteoclasts. Moreover, using Ano5 knockout (KO) mice, we found that they exhibited low bone volume, abnormal calcium deposits, and defective osteoblast and osteoclast differentiation. We also showed that Ano5 deletion in mice significantly diminished [Ca2+]i oscillations in both osteoblasts and osteoclasts, which resulted in reduced WNT/ß-Catenin and RANKL-NFATc1 signaling, respectively. Osteoanabolic treatment of parathyroid hormone was effective in enhancing bone strength in Ano5 KO mice. Consequently, these data demonstrate that Ano5 positively modulates bone homeostasis via calcium signaling in GDD.

Dynamic Characteristics and Predictive Capability of Tumor Voxel Dose-Response Assessed Using 18F-FDG PET/CT Imaging Feedback.

Purpose: Tumor voxel dose-response matrix (DRM) can be quantified using feedback from serial FDG-PET/CT imaging acquired during radiotherapy. This study investigated the dynamic characteristics and the predictive capability of DRM. Methods: FDG-PET/CT images were acquired before and weekly during standard chemoradiotherapy with the treatment dose 2 Gy × 35 from 31 head and neck cancer patients. For each patient, deformable image registration was performed between the pretreatment/baseline PET/CT image and each weekly PET/CT image. Tumor voxel DRM was derived using linear regression on the logarithm of the weekly standard uptake value (SUV) ratios for each tumor voxel, such as SUV measured at a dose level normalized to the baseline SUV0. The dynamic characteristics were evaluated by comparing the DRMi estimated using a single feedback image acquired at the ith treatment week (i = 1, 2, 3, or 4) to the DRM estimated using the last feedback image for each patient. The predictive capability of the DRM estimated using 1 or 2 feedback images was evaluated using the receiver operating characteristic test with respect to the treatment outcome of tumor local-regional control or failure. Results: The mean ± SD of tumor voxel SUV measured at the pretreatment and the 1st, 2nd, 3rd, 4th, and last treatment weeks was 6.76 ± 3.69, 5.72 ± 3.43, 3.85 ± 2.22, 3.27 ± 2.25, 2.5 ± 1.79, and 2.23 ± 1.27, respectively. The deviations between the DRMi estimated using the single feedback image obtained at the ith week and the last feedback image were 0.86 ± 4.87, -0.06 ± 0.3, -0.09 ± 0.17, and -0.09 ± 0.12 for DRM1, DRM2, DRM3, and DRM4, respectively. The predictive capability of DRM3 and DRM4 was significant (p < 0.001). The area under the curve (AUC) was increased with the increase in treatment dose level. The DRMs constructed using the single feedback image achieved an AUC of 0.86~1. The AUC was slightly improved to 0.94~1 for the DRMs estimated using 2 feedback images. Conclusion: Tumor voxel metabolic activity measured using FDG-PET/CT fluctuated noticeably during the first 2 treatment weeks and obtained a stabilized reduction rate thereafter. Tumor voxel DRM constructed using a single FDG-PET/CT feedback image after the 2nd treatment week (>20 Gy) has a good predictive capability. The predictive capability improved continuously using a later feedback image and marginally improved when two feedback images were applied.

Evaluation of DIR schemes on tumor/organ with progressive shrinkage: Accuracy of tumor/organ internal tissue tracking during the radiation treatment.

PURPOSE: Accuracy of intratumoral treatment dose accumulation and response assessment highly depends on the accuracy of a DIR method. However, achievable accuracy of the existing DIR methods for tumor/organ with large and progressive shrinkage during the radiotherapy course have not been explored. This study aimed to use a bio-tissue phantom to quantify the achievable accuracy of different DIR schemes. MATERIALS /METHODS: A fresh porcine liver was used for phantom material. Sixty gold markers were implanted on the surface and inside of the liver. To simulate the progressive radiation-induced tumor/organ shrinkage, the phantom was heated using a microwave oven incrementally from 30 s to 200 s in 8 phases. For each phase, the phantom was scanned by CT. Two extra image sets were generated from the original images: 1) the image set with overriding the high-density gold markers (feature image); 2) the image set with overriding the entire phantom to the mean soft tissue intensity (featureless image). Ten DIR schemes were evaluated to mimic clinical treatment situations of tumor/critical organ with respect to their surface and internal condition of image features, availability of intermediate feedback images and DIR methods. The internal marker's positions were utilized to evaluate DIR accuracy quantified by target registration error (TRE). RESULTS: Volume reduction was about 20 % ∼ 40 % of the initial volume after 90 s âˆ¼ 200 s of the heating. Without image features on the surface and inside of the phantom, the hybrid-DIR (image-based DIR followed by biomechanical model-based refinement) with the surface constraint achieved the registration TRE from 2.6 ± 1.2 mm to 5.3 ± 2.6 mm proportional to the %volume shrinkage. Meanwhile, the hybrid-DIR with the surface-marker constraint achieved the TRE from 2.4 ± 1.2 mm to 2.6 ± 1.0 mm. If both the surface and internal image features would be viable on the feedback images, the achievable accuracy could be minimal with the TRE from 1.6 ± 0.9 mm to 1.9 ± 1.2 mm. CONCLUSIONS: Standard DIR methods cannot guarantee intratumoral tissue registration accuracy for tumor/organ with large progressive shrinkage. Achievable accuracy with using the hybrid DIR method is highly dependent on the surface registration accuracy. If the surface registration mean TRE can be controlled within 2 mm, the mean TRE of internal tissue can be controlled within 3 mm.

Developing an accurate model of spot-scanning treatment delivery time and sequence for a compact superconducting synchrocyclotron proton therapy system.

BACKGROUND: A new compact superconducting synchrocyclotron single-room proton solution delivers pulsed proton beams to each spot through several irradiation bursts calculated by an iterative layer delivery algorithm. Such a mechanism results in a new beam parameter, burst switching time (BST) in the total beam delivery time (BDT) which has never been studied before. In this study, we propose an experimental approach to build an accurate BDT and sequence prediction model for this new proton solution. METHODS: Test fields and clinical treatment plans were used to investigate each beam delivery parameter that impacted BDT. The machine delivery log files were retrospectively analyzed to quantitatively model energy layer switching time (ELST), spot switching time (SSWT), spot spill time (SSPT), and BST. A total of 102 clinical IMPT treatment fields' log files were processed to validate the accuracy of the BDT prediction model in comparison with the result from the current commercial system. Interplay effect is also investigated as a clinical application by comparing this new delivery system model with a conventional cyclotron accelerator model. RESULTS: The study finds that BST depends on the amount of data to be transmitted between two sequential radiation bursts, including a machine irradiation log file of the previous burst and a command file to instruct the proton system to deliver the next burst. The 102 clinical treatment fields showed that the accuracy of each component of the BDT matches well between machine log files and BDT prediction model. More specifically, the difference of ELST, SSWT, SSPT, and BST were (- 3.1 ± 5.7)%, (5.9 ± 3.9)%, (2.6 ± 8.7)%, and (- 2.3 ± 5.3)%, respectively. The average total BDT was about (2.1 ± 3.0)% difference compared to the treatment log files, which was significantly improved from the current commercial proton system prediction (58 ± 15)%. Compared to the conventional cyclotron system, the burst technique from synchrocyclotron effectively reduced the interplay effect in mobile tumor treatment. CONCLUSION: An accurate BDT and sequence prediction model was established for this new clinical compact superconducting synchrocyclotron single-room proton solution. Its application could help users of similar facilities better assess the interplay effect and estimate daily patient treatment throughput.

DZNep promotes mouse bone defect healing via enhancing both osteogenesis and osteoclastogenesis.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression of downstream tumor-suppressing genes. As a small molecular inhibitor of EZH2, 3-Deazaneplanocin (DZNep) has been widely studied due to the role of tumor suppression. With the roles of epigenetic regulation of bone cells emerged in past decades, the property and molecular mechanism of DZNep on enhancing osteogenesis had been reported and attracted a great deal of attention recently. This study aims to elucidate the role of DZNep on EZH2-H3K27me3 axis and downstream factors during both osteoclasts and osteoblasts formation and the therapeutic possibility of DZNep on bone defect healing. METHODS: Bone marrow-derived macrophages (BMMs) cells were cultured, and their responsiveness to DZNep was evaluated by cell counting kit-8, TRAP staining assay, bone resorption assay, podosome actin belt. Bone marrow-derived mesenchymal stem cells (BMSC) were cultured and their responsiveness to DZNep was evaluated by cell counting kit-8, ALP and AR staining assay. The expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Wnt signaling pathway was determined by qPCR and western blotting. Mouse bone defect models were created, rescued by DZNep injection, and the effectiveness was evaluated by X-ray and micro-CT and histological staining. RESULTS: Consistent with the previous study that DZNep enhances osteogenesis via Wnt family member 1(Wnt1), Wnt6, and Wnt10a, our results showed that DZNep also promotes osteoblasts differentiation and mineralization through the EZH2-H3K27me3-Wnt4 axis. Furthermore, we identified that DZNep promoted the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation via facilitating the phosphorylation of IKKα/ß, IκB, and subsequently NF-κB nuclear translocation, which credit to the EZH2-H3K27me3-Foxc1 axis. More importantly, the enhanced osteogenesis and osteoclastogenesis result in accelerated mice bone defect healing in vivo. CONCLUSION: DZNep targeting EZH2-H3K27me3 axis facilitated the healing of mice bone defect via simultaneously enhancing osteoclastic bone resorption and promoting osteoblastic bone formation.

Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair.

Rationale: Angiogenesis and osteogenesis are highly coupled processes which are indispensable to bone repair. However, the underlying mechanism(s) remain elusive. To bridge the gap in understanding the coupling process is crucial to develop corresponding solutions to abnormal bone healing. Epidermal growth factor-like protein 6 (EGFL6) is an angiogenic factor specifically and distinctively up-regulated during osteoblast differentiation. In contrast with most currently known osteoblast-derived coupling factors, EGFL6 is highlighted with little or low expression in other cells and tissues. Methods: In this study, primary bone marrow mesenchymal stem cells (MSCs) and osteoblastic cell line (MC3T3-E1) were transduced with lentiviral silencing or overexpression constructs targeting EGFL6. Cells were induced by osteogenic medium, followed by the evaluation of mineralization as well as related gene and protein expression. Global and conditional knockout mice were established to examine the bone phenotype under physiological condition. Furthermore, bone defect models were created to investigate the outcome of bone repair in mice lacking EGFL6 expression. Results: We show that overexpression of EGFL6 markedly enhances osteogenic capacity in vitro by augmenting bone morphogenic protein (BMP)-Smad and MAPK signaling, whereas downregulation of EGFL6 diminishes osteoblastic mineralization. Interestingly, osteoblast differentiation was not affected by the exogenous addition of EGFL6 protein, thereby indicating that EGFL6 may regulate osteoblastic function in an intracrine manner. Mice with osteoblast-specific and global knockout of EGFL6 surprisingly exhibit a normal bone phenotype under physiological conditions. However, EGFL6-deficiency leads to compromised bone repair in a bone defect model which is characterized by decreased formation of type H vessels as well as osteoblast lineage cells. Conclusions: Together, these data demonstrate that EGFL6 serves as an essential regulator to couple osteogenesis to angiogenesis during bone repair.

Long Non-coding RNA RP11-395G23.3 Acts as a Competing Endogenous RNA of miR-124-3p to Regulate ROR1 in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human malignancies with poor prognosis. However, the underlying mechanisms of ATC remain to be elucidated. Recently, increasing studies have focused on competitive endogenous RNA (ceRNA) to discover valuable biomarkers for the diagnosis of ATC. The present study identified 705 differentially expressed mRNAs and 47 differentially expressed lncRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were also conducted. Additionally, an lncRNA/miRNA/mRNA network was constructed which included 1103 regulatory relations. The upregulation of RP11-395G23.3 in ATC cells was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In the loss of function assays, results suggested silencing of RP11-395G23.3 inhibited cell proliferation and induced cell apoptosis. Mechanically, RP11-395G23.3 could increase ROR1 via sponging miR-124-3p as a ceRNA. Moreover, ROR1 expression was decreased with the downregulation of RP11-395G23.3, but was rescued by the co-transfection of the miR-124-3p inhibitor in ATC cells. Our research suggested that the RP11-395G23.3/miR-124-3p/ROR1 axis potentially acted as a potential target for the diagnosis of ATC.

Combination of AZD3463 and DZNep Prevents Bone Metastasis of Breast Cancer by Suppressing Akt Signaling.

Osteolysis resulting from osteoclast overactivation is one of the severe complications of breast cancer metastasis to the bone. Previous studies reported that the anti-cancer agent DZNep induces cancer cell apoptosis by activating Akt signaling. However, the effect of DZNep on breast cancer bone metastasis is unknown. We previously found that DZNep enhances osteoclast differentiation by activating Akt. Therefore, we explored the use of the anti-cancer agent AZD3463 (an Akt inhibitor) along with DZNep, as AZD3463 can act as an anti-cancer agent and can also potentially ameliorate bone erosion. We evaluated osteoclast and breast cancer cell phenotypes and Akt signaling in vitro by treating cells with DZNep and AZD3463. Furthermore, we developed a breast cancer bone metastasis animal model in mouse tibiae to further determine their combined effects in vivo. Treatment of osteoclast precursor cells with DZNep alone increased osteoclast differentiation, bone resorption, and expression of osteoclast-specific genes. These effects were ameliorated by AZD3463. The combination of DZNep and AZD3463 inhibited breast cancer cell proliferation, colony formation, migration, and invasion. Finally, intraperitoneal injection of DZNep and AZD3463 ameliorated tumor progression and protected against bone loss. In summary, DZNep combined with AZD3463 prevented skeletal complications and inhibited breast cancer progression by suppressing Akt signaling.