Connexin 36 Mediated Intercellular Endoplasmic Reticulum Stress Transmission Induces SSTA Resistance in Growth Hormone Pituitary Adenoma.

Somatostatin analogues (SSTA) are first-line pharmacological treatment choice for acromegaly, which received satisfying tumor shrinkage and normalization of growth hormone. However, there are still patients unresponsive to SSTA, and the underline mechanism remains unknown. Besides, there is no evidence regarding the role of endoplasmic reticulum stress (ERS) and its transmission in SSTA resistance, which also require investigation. Primary growth hormone adenoma cells and cell lines were treated with SSTA; autophagy double-labeled LC3 (mRFP-GFP) adenovirus transfection, flow cytometry sorting, western blotting, calcium imaging as well as immunofluorescence staining were used to determine ERS and autophagy signal transmission; xenograft and syngeneic tumor in vivo model were exploited to confirm the ERS signal transmission mediated effect. Our results revealed that SSTA induces ERS in pituitary growth hormone (GH) adenoma cells. The ERS signals can be intercellularly transmitted, leading to less responsible to SSTA treatment. Moreover, SSTA stimulates inositol triphosphate (IP3) elevation, mediating ERS intercellular transfer. In addition, connexin 36 tunnels ERS transmission, and its blocker, Quinine, exhibits a synergistic effect with SSTA treating GH adenoma. Our study provided insight into ERS intercellular transmission mediated SSTA resistance, which could be translated into clinical usage to improve SSTA efficiency in GH adenoma treatment.

The miR-19a/Cylindromatosis Axis Regulates Pituitary Adenoma Bone Invasion by Promoting Osteoclast Differentiation.

BACKGROUND: The presence of bone invasion in aggressive pituitary adenoma (PA) was found in our previous study, suggesting that PA cells may be involved in the process of osteoclastogenesis. miR-19a (as a key member of the miR-17-92 cluster) has been reported to activate the nuclear factor-кB (NF-кB) pathway and promote inflammation, which could be involved in the process of the bone invasion of pituitary adenoma. METHODS: In this work, FISH was applied to detect miR-19a distribution in tissues from patients with PA. A model of bone invasion in PA was established, GH3 cells were transfected with miR-19a mimic, and the grade of osteoclastosis was detected by HE staining. qPCR was performed to determine the expression of miR-19a throughout the course of RANKL-induced osteoclastogenesis. After transfected with a miR-19a mimic, BMMs were treated with RANKL for the indicated time, and the osteoclast marker genes were detected by qPCR and Western Blot. Pit formation and F-actin ring assay were used to evaluate the function of osteoclast. The TargetScan database and GSEA were used to find the potential downstream of miR-19a, which was verified by Co-IP, Western Blot, and EMSA. RESULTS: Here, we found that miR-19a expression levels were significantly correlated with the bone invasion of PA, both in clinical samples and animal models. The osteoclast formation prior to bone resorption was dramatically enhanced by miR-19, which was mediated by decreased cylindromatosis (CYLD) expression, increasing the K63 ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6). Consequently, miR-19a promotes osteoclastogenesis by the activation of the downstream NF-кB and mitogen-activated protein kinase (MAPK) pathways. CONCLUSIONS: To summarize, the results of this study indicate that PA-derived miR-19a promotes osteoclastogenesis by inhibiting CYLD expression and enhancing the activation of the NF-кB and MAPK pathways.

A disintegrin and metalloproteinase 22 activates integrin ß1 through its disintegrin domain to promote the progression of pituitary adenoma.

BACKGROUND: Approximately 35% of pituitary adenoma (PA) display an aggressive profile, resulting in low surgical total resection rates, high recurrence rates, and worse prognosis. However, the molecular mechanism of PA invasion remains poorly understood. Although "a disintegrin and metalloproteinases" (ADAMs) are associated with the progression of many tumors, there are no reports on ADAM22 in PA. METHODS: PA transcriptomics databases and clinical specimens were used to analyze the expression of ADAM22. PA cell lines overexpressing wild-type ADAM22, the point mutation ADAM22, the mutated ADAM22 without disintegrin domain, and knocking down ADAM22 were generated. Cell proliferation/invasion assays, flow cytometry, immunohistochemistry, immunofluorescence, co-immunoprecipitation, mass spectrometry, Reverse transcription-quantitative real-time PCR, phos-tag SDS-PAGE, and Western blot were performed for function and mechanism research. Nude mice xenograft models and rat prolactinoma orthotopic models were used to validate in vitro findings. RESULTS: ADAM22 was significantly overexpressed in PA and could promote the proliferation, migration, and invasion of PA cells. ADAM22 interacted with integrin ß1 (ITGB1) and activated FAK/PI3K and FAK/ERK signaling pathways through its disintegrin domain to promote PA progression. ADAM22 was phosphorylated by PKA and recruited 14-3-3, thereby delaying its degradation. ITGB1-targeted inhibitor (anti-itgb1) exerted antitumor effects and synergistic effects in combination with somatostatin analogs or dopamine agonists in treating PA. CONCLUSIONS: ADAM22 was upregulated in PA and was able to promote PA proliferation, migration, and invasion by activating ITGB1 signaling. PKA may regulate the degradation of ADAM22 through post-transcriptional modification levels. ITGB1 may be a potential therapeutic target for PA.

Tumor-Associated Fibroblast-Derived Exosomal circDennd1b Promotes Pituitary Adenoma Progression by Modulating the miR-145-5p/ONECUT2 Axis and Activating the MAPK Pathway.

TAF participated in the progression of various cancers, including PA via the release of soluble factors. Exosomes belonged to extracellular vesicles, which were revealed as a crucial participator in intercellular communication. However, the expression pattern and effect of TAF-derived exosomes remained largely unknown in PA. In the present study, we performed in silico analysis based on public RNA-seq datasets to generate the circRNA/miRNA regulatory network. The qRT-PCR, Western blotting, RNA pull-down, and luciferase assay were performed to investigate the effect of TAF-derived exosomes. TAF-derived exosomal circDennd1b was significantly upregulated in PA and promoted the proliferation, migration, and invasion of PA cells via sponging miR-145-5p in PA cells. In addition, miR-145-5p directly regulated One Cut homeobox 2 (ONECUT2/OC2) expression and inhibited the promoting effect of ONECUT2 on PA. We further demonstrated that ONECUT2 transcriptionally increased fibroblast growth factor receptor 3 (FGFR3) expression, which further activates the mitogen-activated protein kinases (MAPK) pathway, thus promoting PA progression. Moreover, the suppression of TAFs by ABT-263 and ONECUT2 by CSRM617 inhibited the growth of PA. In conclusion, our study illustrated that TAF-derived exosomal circDennd1b affected PA progression via regulating ONECUT2 expression, which provides a potential therapeutic strategy against aggressive PA.

PKCθ Regulates Pituitary Adenoma Bone Invasion by Activating Osteoclast in NF-κB/IL-1ß-Dependent Manner.

BACKGROUND: Pituitary adenoma (PA) bone invasion results in adverse outcomes, such as reduced rates of complete surgical resection and biochemical remission as well as increased recurrence rates, though few studies have been conducted. METHODS: We collected clinical specimens of PAs for staining and statistical analysis. Evaluation of the ability of PA cells to induce monocyte-osteoclast differentiation by coculturing PA cells with RAW264.7 in vitro. An in vivo model of bone invasion was used to simulate the process of bone erosion and evaluate the effect of different interventions in alleviating bone invasion. RESULTS: We found an overactivation of osteoclasts in bone-invasive PAs and concomitant aggregation of inflammatory factors. Furthermore, activation of PKCθ in PAs was established as a central signaling promoting PA bone invasion through the PKCθ/NF-κB/IL-1ß pathway. By inhibiting PKCθ and blocking IL1ß, we were able to significantly reverse bone invasion in an in vivo study. Meanwhile, we also found that celastrol, as a natural product, can obviously reduce the secretion of IL-1ß as well as alleviate the progression of bone invasion. CONCLUSIONS: By activating the PKCθ/NF-κB/IL-1ß pathway, pituitary tumors are able to induce monocyte-osteoclast differentiation in a paracrine manner and promote bone invasion, which can be alleviated by celastrol.

Exploiting D2 receptor ß-arrestin2-biased signalling to suppress tumour growth of pituitary adenomas.

BACKGROUND AND PURPOSE: Dopamine agonists targeting D2 receptor have been used for decades in treating pituitary adenomas. There has been little clear evidence implicating the canonical G protein signalling as the mechanism by which D2 receptor suppresses the growth of pituitary tumours. We hypothesize that ß-arrestin2-dependent signalling is the molecular mechanism dictating D2 receptor inhibitory effects on pituitary tumour growth. EXPERIMENTAL APPROACH: The involvement of G protein and ß-arrestin2 in bromocriptine-mediated growth suppression in rat MMQ and GH3 tumour cells was assessed. The anti-growth effect of a ß-arrestin2-biased agonist, UNC9994, was tested in cultured cells, tumour-bearing nude mice and primary cultured human pituitary adenomas. The effect of G protein signalling on tumour growth was also analysed by using a G protein-biased agonist, MLS1547, and a Gßγ inhibitor, gallein, in vitro. KEY RESULTS: ß-arrestin2 signalling but not G protein pathways mediated the suppressive effect of bromocriptine on pituitary tumour growth. UNC9994 inhibited pituitary tumour cell growth in vitro and in vivo. The suppressive function of UNC9994 was obtained by inducing intracellular reactive oxygen species generation through downregulating mitochondrial complex I subunit NDUFA1. The effects of Gαi/o signalling and Gßγ signalling via D2 receptor on pituitary tumour growth were cell-type-dependent. CONCLUSION AND IMPLICATIONS: Given the very low expression of Gαi/o proteins in pituitary tumours and the complexity of the responses of pituitary tumours to G protein signalling pathways, our study reveals D2 receptor ß-arrestin2-biased ligand may be a more promising choice to treat pituitary tumours with improved therapeutic selectivity.

Serum- and Glucocorticoid-inducible Kinase 1 is Essential for Osteoclastogenesis and Promotes Breast Cancer Bone Metastasis.

Bone metastasis is a severe complication associated with various carcinomas. It causes debilitating pain and pathologic fractures and dramatically impairs patients' quality of life. Drugs aimed at osteoclast formation significantly reduce the incidence of skeletal complications and are currently the standard treatment for patients with bone metastases. Here, we reported that serum- and glucocorticoid-inducible kinase 1 (SGK1) plays a pivotal role in the formation and function of osteoclasts by regulating the Ca2+ release-activated Ca2+ channel Orai1. We showed that SGK1 inhibition represses osteoclastogenesis in vitro and prevents bone loss in vivo Furthermore, we validated the effect of SGK1 on bone metastasis by using an intracardiac injection model in mice. Inhibition of SGK1 resulted in a significant reduction in bone metastasis. Subsequently, the Oncomine and the OncoLnc database were employed to verify the differential expression and the association with clinical outcome of SGK1 gene in patients with breast cancer. Our data mechanistically demonstrated the regulation of the SGK1 in the process of osteoclastogenesis and revealed SGK1 as a valuable target for curing bone metastasis diseases.

Co-Expression Network Analysis Identified Gene Signatures in Osteosarcoma as a Predictive Tool for Lung Metastasis and Survival.

Osteosarcoma (OS) is the most common primary bone tumor, whose poor prognosis is mainly due to lung metastasis. The aim of this study is to build a practical and valid diagnostic test that can predict the risk of OS metastasis and progression. We performed weighted gene co-expression network analysis (WGCNA) on GSE21257 from the Gene Expression Omnibus (GEO) database, which contains microarray data of biopsies from OS patients. In these modules, the highest association was found between the blue module and metastasis stage (r = -0.52) by Pearson's correlation analysis. Based on Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression, we derived eight clinically significant genes and constructed an eight-gene signature for metastasis status. It showed great efficacy to distinguish metastasis from non-metastasis (AUC = 0.886) and the results were validated in The Cancer Genome Atlas (TCGA) database. Functional enrichment analysis of hub genes showed that their biological processes focused on immune-related pathways, suggesting the important roles of immune cells, immune pathways and the tumor microenvironment in metastasis development. In conclusion, we discovered an efficient gene signature with great efficacy to distinguish metastasis status, which may help improve early diagnosis and treatment, enhancing the clinical outcomes of OS patients. Besides we created an effective protocol to seek for several hub genes in high-throughput data by combining WGCNA and LASSO Cox regression.

Identification and characterization of microglia/macrophages in the granuloma microenvironment of encephalic schistosomiasis japonicum.

BACKGROUND: Egg-induced immune response and granuloma formation are thought to be the basis of central nervous system (CNS)-related clinical symptoms of Schistosoma japonicum. Microglia/macrophages are the major immune cells involved in detection and subsequent elimination of pathogens and injured tissue in the brain. However, little is known about their role in the pathogenesis of neuroschistosomiasis. The main purpose of the study is to clarify the pathological involvement of microglia/macrophages in the pathogenesis of neuroschistosomiasis (NS). METHODS: Staining techniques were applied to the granuloma tissues excised from 4 patients, as well as mice model which was established by microinjecting viable S. japonicum eggs into the brain. Clinical features of the patients and neurological symptoms in mice were also collected and analyzed in terms of their correlation with microglia/macrophages. RESULTS: Microglia/macrophages constituted the major portions of the granulomas surrounding the eggs in both all human cases and S. japonicum egg-injected mice. Granuloma persisted in all patients accompanied by unremitted neurological symptoms, while in mice granuloma formation initiated on day 3, peaked on day 7 and subsided on day 30 post injection with S. japonicum eggs. No neurological abnormalities were observed in egg-injected mice except for significant weight decrease on day 3 compared with saline-injected control. M1 polarization of microglia/macrophages was confirmed in egg-injected mice 3 days post injection and in all human cases. M2 polarization was absent in human patients despite the duration of complaints but dominated in the whole progression of egg-induced pathology in mice until the elimination of eggs and subsidence of neuroinflammation on day 30 post injection. CONCLUSIONS: Microglia/macrophages participated actively in the granuloma microenvironment of encephalic schistosomiasis japonicum in both human and mice. The polarization pattern of microglia/macrophages coincided with the symptomatic features in human cases and S. japonicum egg-injected mice, indicating M2 instead of M1 activation as a probably more important mediator in the battle against egg-induced pathology and concomitant manifestations. These new findings will shed light on the pathogenesis of NS from a brand-new perspective, and may contribute to the immunotherapy development for such disease, favoring perhaps M2 polarization of microglia/macrophages as a feasible strategy.

Clinical Characteristics, Imaging Findings and Surgical Outcomes of Chiari Malformation Type I in Pediatric and Adult Patients.

A growing number of children and adolescents are being diagnosed as Chiari malformation type I (CM-I) for behavioral disorders, developmental delay, seizures, or abnormal orpharyngeal function. The aim of this study was to compare the clinical characteristics, imaging findings and surgical outcomes of CM-I in pediatric and adult patients. Between January 2014 and June 2017, 84 patients with CM-I underwent surgical treatment in our department. We divided the patients into two groups: pediatric group (n=11, age <18 years) and adult group (n=73, age ≥18 years). Data on clinical characteristics, imaging findings, surgical outcomes, and prognosis were retrospectively reviewed and compared between these two groups. For clinical presentation, scoliosis (36.4%) and developmental delay (36.4%) were more common in pediatric patients, whereas, sensory disturbance (58.9%) and motor weakness (41.1%) were more common in adult patients. Imaging findings showed that the incidence of hydrocephalus and craniovertebral junctional abnormalities was significantly higher in pediatric group than in adult group (P<0.05). Compared to adult group, pediatric group showed a better improvement or resolution of syrinx and tonsillar herniation after surgical treatments (P<0.05). The total Chicago Chiari Outcome Scale (CCOS) score in pediatric patients at the last followup was significantly higher than that in adult patients (P=0.002). In conclusion, the clinical characteristics and imaging findings appeared to be different in pediatric and adult patients with CM-I. The surgical outcomes of pediatric patients were shown to be significantly better than those of adult patients.

PKCδ reveals a tumor promoter function by promoting cell proliferation and migration in somatotropinomas.

Protein kinase C δ (PKCδ), a subtype of PKC family, has been recognized as a tumor promoter or suppressor depending on different tissue specificities in various tumor types. However, the effects of PKCδ on somatotropinomas are poorly understood. This study aims to explore the precise role of PKCδ in promoting tumor progression in somatotropinomas. In the present study, we examined the expression levels of PKCδ in clinical specimens of human somatotropinomas to show that PKCδ overexpression correlated with invasive properties of somatotropinomas. Furthermore, we employed rat anterior pituitary GH3 cells as the experiment model to demonstrate that PKCδ activation by PKC agonist (Phorbol-12-myristate-13-acetate, PMA) significantly promoted the proliferation and migration potential of GH3 cells, and these effects could be abolished following PKCδ inhibition by specific inhibitor Rottlerin. Mechanistically, PKCδ activated ERK1/2 signaling, which was responsible for PKCδ-induced promotion of GH3 cell proliferation and migration. Taken together, our results indicated that PKCδ functions as a tumor promoter by promoting cell proliferation and migration in somatotropinomas.