DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long noncoding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo disease models. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, which indicates mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease.

DNAJB1-PRKACA fusion neoantigens elicit rare endogenous T cell responses that potentiate cell therapy for fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 T cell responses to this fusion in FLC patients and evaluate fusion-specific T cell receptors (TCRs) for use in cellular immunotherapies. We observe that fusion-specific CD8 T cells are rare and that FLC patient TCR repertoires lack large clusters of related TCR sequences characteristic of potent antigen-specific responses, potentially explaining why endogenous immune responses are insufficient to clear FLC tumors. Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity in vivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC.

The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a worldwide threat in the past 3 years. Although it has been widely and intensively investigated, the mechanism underlying the coronavirus-host interaction requires further elucidation, which may contribute to the development of new antiviral strategies. Here, we demonstrated that the host cAMP-responsive element-binding protein (CREB1) interacts with the non-structural protein 13 (nsp13) of SARS-CoV-2, a conserved helicase for coronavirus replication, both in cells and in lung tissues subjected to SARS-CoV-2 infection. The ATPase and helicase activity of viral nsp13 were shown to be potentiated by CREB1 association, as well as by Protein kinase A (PKA)-mediated CREB1 activation. SARS-CoV-2 replication is significantly suppressed by PKA Cα, cAMP-activated protein kinase catalytic subunit alpha (PRKACA), and CREB1 knockdown or inhibition. Consistently, the CREB1 inhibitor 666-15 has shown significant antiviral effects against both the WIV04 strain and the Omicron strain of the SARS-CoV-2. Our findings indicate that the PKA-CREB1 signaling axis may serve as a novel therapeutic target against coronavirus infection. IMPORTANCE: In this study, we provide solid evidence that host transcription factor cAMP-responsive element-binding protein (CREB1) interacts directly with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) helicase non-structural protein 13 (nsp13) and potentiate its ATPase and helicase activity. And by live SARS-CoV-2 virus infection, the inhibition of CREB1 dramatically impairs SARS-CoV-2 replication in vivo. Notably, the IC50 of CREB1 inhibitor 666-15 is comparable to that of remdesivir. These results may extend to all highly pathogenic coronaviruses due to the conserved nsp13 sequences in the virus.

GalNAc-conjugated siRNA targeting the DNAJB1-PRKACA fusion junction in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). Current therapies such as chemotherapy and radiation have limited efficacy, and new treatment options are needed urgently. We have previously shown that FLC tumors are dependent on the fusion kinase DNAJB1::PRKACA, making the oncokinase an ideal drug target. mRNA degrading modalities such as antisense oligonucleotides or small interfering RNAs (siRNAs) provide an opportunity to specifically target the fusion junction. Here, we identify a potent and specific siRNA that inhibits DNAJB1::PRKACA expression. We found expression of the asialoglycoprotein receptor in FLC to be maintained at sufficient levels to effectively deliver siRNA conjugated to the GalNAc ligand. We observe productive uptake and siRNA activity in FLC patient-derived xenografts (PDX) models in vitro and in vivo. Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.

Gene Rearrangement and Expression of PRKACA and PRKACB Govern Morphobiology of Pancreatobiliary Oncocytic Neoplasms.

Intraductal oncocytic papillary neoplasms (IOPNs) are distinct from intraductal papillary mucinous neoplasms based on characteristic morphologic and genetic features represented by fusion genes involving PRKACA or PRKACB (PRKACA/B). However, pancreatic and biliary tumors with partial oncocytic features are often encountered clinically, and their molecular features are yet to be clarified. This study included 80 intraductal papillary neoplasms: 32 tumors with mature IOPN morphology (typical), 28 with partial or subclonal oncocytic features (atypical), and 20 without oncocytic features (control). We analyzed PRKACA/B fusion genes, including ATP1B1::PRKACA, DNAJB1::PRKACA, and ATP1B1::PRKACB, by reverse-transcription PCR; mRNA expression of fusion genes and nonrearranged PRKACA/B genes by quantitative reverse-transcription PCR; mutations in KRAS, BRAF, and GNAS by targeted sequencing or droplet digital PCR; and the expression of cyclic adenosine monophosphate (cAMP)-dependent protein kinase catalytic subunits α (PRKACA) and ß (PRKACB), phosphorylated cAMP response element-binding protein, and aberrations of p16, p53, SMAD4, STK11, and ß-catenin by immunohistochemistry. PRKACA/B fusion genes were detected in 100% (32/32) of typical, 46% (13/28) of atypical, and 0% (0/20) of control (P < .05). Expression of PRKACA, PRKACB, and phosphorylated cAMP response element-binding protein was upregulated in neoplasms with PRKACA/B fusion genes (P < .05). mRNA expression of the PRKACA/B fusion genes and protein expression of PRKACA or PRKACB tended to be higher in typical than in atypical cases (mRNA, P = .002; protein expression, P = .054). In some atypical neoplasms with mixed subtypes, PRKACA/B fusion genes were superimposed exclusively on oncocytic components. Typical IOPNs harbored fewer KRAS and GNAS mutations than control samples and fewer alterations in p53 and STK11 than atypical samples (P < .05). In conclusion, PRKACA/B fusion genes not only are the characteristic drivers of IOPNs but also play a crucial role in the development of subclonal oncocytic neoplasms. Moreover, oncocytic morphology is strongly associated with upregulation of PRKACA/B, which may provide clues for potential therapeutic options.

Phoenixin-20 ameliorates Sevoflurane inhalation-induced post-operative cognitive dysfunction in rats via activation of the PKA/CREB signaling.

Post-operative cognitive dysfunction (POCD) is a common complication after surgery due to the usage of anesthetics, such as Sevoflurane, which severely impacts the life quality of patients. Currently, the pathogenesis of Sevoflurane-induced POCD has not been fully elucidated but is reportedly involved with oxidative stress (OS) injury and aggravated inflammation. Phoenixin-20 (PNX-20) is a PNX peptide consisting of 20 amino acids with promising inhibitory effects on OS and inflammation. Herein, we proposed to explore the potential protective function of PNX-20 on Sevoflurane inhalation-induced POCD in rats. Sprague-Dawley (SD) rats were treated with 100 ng/g PNX-20 for 7 days with or without pre-inhalation with 2.2% Sevoflurane. Markedly increased escape latency and decreased time in the target quadrant in the Morris water maze (MWM) test, and aggravated pathological changes and apoptosis in the hippocampus tissue were observed in Sevoflurane-treated rats, which were markedly attenuated by PNX-20. Furthermore, the aggravated inflammation and OS in the hippocampus observed in Sevoflurane-treated rats were notably abolished by PNX-20. Moreover, the brain-derived neurotrophic factor (BDNF), protein kinase A (PKA), and phospho-cAMP response element binding protein/cAMP response element binding protein (p-CREB/CREB) levels were markedly decreased in Sevoflurane-treated rats, which were memorably increased by PNX-20. Our results indicated that PNX-20 ameliorated Sevoflurane inhalation-induced POCD in rats via the activation of PKA/CREB signaling, which might supply a new treatment approach for POCD.

Germline PRKACA amplification-associated primary pigmented nodular adrenocortical disease: a case report and literature review.

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare adrenocorticotropin hormone (ACTH)-independent Cushing's syndrome (CS). Pediatric patients with PPNAD typically have unusual skin lesions and slow growth with unknown causes. We present a case of a female Chinese patient with PPNAD caused by the germline PRKACA gene copy number gain of chromosome 19. The patient initially presented with kidney stones, short stature, and obesity. After further testing, it was discovered that the patient had diabetes, mild hypertension, low bone mass, a low ACTH level, and hypercortisolemia, and neither the low-dose or high-dose dexamethasone suppression test was able to inhibit hematuric cortisol, which paradoxically increased. PPNAD was pathologically diagnosed after unilateral adrenalectomy. Chromosome microarrays and whole exon sequencing analyses of the peripheral blood, as well as testing of sectioned adrenal tissue, showed a rise in the copy number of the duplication-containing PRKACA gene on chromosome 19p13.13p13.12, a de novo but not heritable gene defect that causes disease. The clinical signs and symptoms supported the diagnosis of Carney complex (CNC). One significant mechanism of CNC pathogenesis may be the rise in germline PRKACA copy number of chromosome 19. When assessing PPNAD patients for CNC, the possibility of PRKACA gene amplification should be considered. The effect of PRKACA gene amplification on the clinical manifestations of CNC needs to be confirmed by more cases.

Oncogenic Addiction of Fibrolamellar Hepatocellular Carcinoma to the Fusion Kinase DNAJB1-PRKACA.

PURPOSE: Gene fusions are drivers of many pediatric tumors. In fibrolamellar hepatocellular carcinoma (FLC), a fusion of DNAJB1 and PRKACA is the dominant recurrent mutation. Expression of the DNAJB1-PRKACA fusion gene in mice results in a tumor that recapitulates FLC. However, it is not known whether transient expression of DNAJB1-PRKACA is sufficient only to trigger tumor formation or whether ongoing expression is necessary for maintenance and progression. EXPERIMENTAL DESIGN: We screened short hairpin RNAs (shRNA) tiled over the fusion junction and identified several potent and specific candidates in vitro and two independent FLC patient-derived xenografts (PDX). RESULTS: We show that continued DNAJB1-PRKACA expression is not only required for continued tumor growth, but additionally its inhibition results in cell death. Inhibition of DNAJB1-PRKACA by an inducible shRNA in cells of PDX of FLC resulted in cell death in vitro. Induction of the shRNA inhibits FLC tumors growing in mice with no effect on xenografts from a hepatocellular carcinoma cell line engineered to express DNAJB1-PRKACA. CONCLUSIONS: Our results validate DNAJB1-PRKACA as the oncogene in FLC and demonstrate both a continued requirement for the oncogene for tumor growth as well as an oncogenic addiction that can be exploited for targeted therapies. We anticipate our approach will be useful for investigations of other fusion genes in pediatric cancers and spur development of precision therapies.

miR-200a-3p Regulates PRKACB and Participates in Aluminium-Induced Tau Phosphorylation in PC12 Cells.

Aluminium (Al) is an environmental neurotoxin that humans are widely exposed to, but the molecular mechanism of its toxic effects is not fully understood. Many studies have shown that exposure to Al can cause abnormal phosphorylation of the tau protein that is believed as one of pathological features of Alzheimer's disease. Increasing evidence indicates that microRNAs (miRNAs) may be involved in the pathological processes of neurodegenerative diseases and are potential regulatory factors for related target genes. Phosphorylation at Ser-133 of cAMP response element-binding protein (CREB) is one of the major pathways of CREB activation, and phosphorylation at this site is controlled by protein kinase A (PKA). The catalytic subunit of PKA, cAMP-dependent protein kinase catalytic subunit beta (PRKACB), phosphorylates CREB. The target gene prediction software TargetScan showed that PRKACB was one of the target mRNAs of miR-200a-3p. The purpose of this study was to investigate whether miR-200a-3p regulates the PKA/CREB pathway by targeting PRKACB and leads to abnormal phosphorylation of the tau protein in nerve cells. The results showed that Al exposure increased the expression level of miR-200a-3p, and miR-200a-3p increased the expression of targeted downregulated PRKACB, and then decreased the PKA/CREB signalling pathway activity, leading to abnormal hyperphosphorylation of tau.

Vascular and Liver Homeostasis in Juvenile Mice Require Endothelial Cyclic AMP-Dependent Protein Kinase A.

During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1-P3) of later (P28-P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKAiEC mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program.

The oncogenic fusion protein DNAJB1-PRKACA can be specifically targeted by peptide-based immunotherapy in fibrolamellar hepatocellular carcinoma.

The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease lacking specific therapies. This study reports on the identification, characterization, and immunotherapeutic application of HLA-presented neoantigens specific for the DNAJB1-PRKACA fusion transcript in fibrolamellar hepatocellular carcinoma. DNAJB1-PRKACA-derived HLA class I and HLA class II ligands induce multifunctional cytotoxic CD8+ and T-helper 1 CD4+ T cells, and their cellular processing and presentation in DNAJB1-PRKACA expressing tumor cells is demonstrated by mass spectrometry-based immunopeptidome analysis. Single-cell RNA sequencing further identifies multiple T cell receptors from DNAJB1-PRKACA-specific T cells. Vaccination of a fibrolamellar hepatocellular carcinoma patient, suffering from recurrent short interval disease relapses, with DNAJB1-PRKACA-derived peptides under continued Poly (ADP-ribose) polymerase inhibitor therapy induces multifunctional CD4+ T cells, with an activated T-helper 1 phenotype and high T cell receptor clonality. Vaccine-induced DNAJB1-PRKACA-specific T cell responses persist over time and, in contrast to various previous treatments, are accompanied by durable relapse free survival of the patient for more than 21 months post vaccination. Our preclinical and clinical findings identify the DNAJB1-PRKACA protein as source for immunogenic neoepitopes and corresponding T cell receptors and provide efficacy in a single-patient study of T cell-based immunotherapy specifically targeting this oncogenic fusion.

Investigation of -PRKACA/-PRKACB fusion genes in oncocytic tumors of the pancreatobiliary and other systems.

Intraductal oncocytic papillary neoplasms (IOPNs) of the pancreatobiliary system are tumors comprising oncocytic cells, in which three types of fusion genes involving -PRKACA/-PRKACB were recently identified. IOPNs infrequently combine with other histological subtypes of pancreatic intraductal papillary mucinous neoplasms (IPMNs) and intraductal papillary neoplasms of the bile duct (IPNBs). This study aimed to confirm the sensitivity/specificity of the fusion genes for IOPNs and to examine their significance in other oncocytic lesions. An RT-PCR, followed by DNA sequencing, was undertaken to examine the fusions in 18 histologically diagnosed IOPNs, including four combined IOPNs. Moreover, in two IOPN cases, invasive carcinomatous lesions were separately examined on their fusion status. Oncocytic thyroidal (n = 10), renal (n = 10), and salivary gland (n = 3) lesions and IPMNs (n = 9)/IPNBs (n = 4) with focal oncocytic changes were examined as controls. Fluorescence in situ hybridization using PRKACA break-apart probes was conducted for the combined IOPN cases. Target sequencing of KRAS exon2/3 and GNAS exon 8/9 was performed for IOPN cases. Fusions were detected in all IOPN cases including invasive lesions/none of the control cases. The fusion event was confirmed also in non-IOPN component in one of the four combined cases. Regarding mutation events, 5.6%/0% of IOPNs were KRAS-mt/GNAS-mt, respectively, and both components of combined IOPNs were all KRAS-wt/GNAS-wt. In conclusion, our study confirmed the sensitivity and specificity of these fusions for IOPNs. Here, we analyzed the roles of these fusion genes in combined IOPNs, proposing the possibility of IOPN development via IPMNs/IPNBs. Further studies with more combined cases are warranted.

The Use of Fluorescence in situ Hybridization to Confirm PRKACA Gene Rearrangement in Fibrolamellar Hepatocellular Carcinoma: A Validation Study.

OBJECTIVE: The objectives of this study are to define the specificity of the DNAJB1-PRKACA fusion transcript for the fibrolamellar subtype of hepatocellular carcinoma (FL-HCC) by testing a targeted sampling of other hepatic neoplasms/proliferations and extrahepatic neoplasms seen in children and young adults and to develop a FISH assay using a commercially available PRKACA break apart probe for use in a CLIA-certified clinical laboratory. METHODS: Formalin fixed paraffin embedded tissue sections from 12 FL-HCC cases, 142 cases of other hepatic neoplasms/proliferations (conventional HCC, focal nodular hyperplasia (FNH), hepatocellular adenoma (HA) and hepatoblastoma (HB)) and extrahepatic neoplasms (neuroblastoma (NB), Wilms tumor (WT) and Gastrointestinal neuroendocrine tumor (GNET)) and 60 matched background normal control tissues underwent fluorescence in situ hybridization (FISH) testing using a break apart probe targeting the PRKACA gene locus on chromosome 19 using standard techniques. RESULTS: The PRKACA gene rearrangement was detected in 11/12 (92%) FL-HCC cases and 1/94 (1%) of conventional HCC cases. All other cases and background control tissues were negative for the PRKACA gene rearrangement. These findings establish a test sensitivity of 91.7% and specificity of 99.5%. CONCLUSION: This study shows that, using standard techniques, FISH testing with a commercially available break apart probe targeting the PRKACA gene can be used as a surrogate for the DNAJB1-PRKACA fusion commonly found in FL-HCC. Also, the PRKACA gene rearrangement is not expressed in other hepatic neo-plasms/proliferations or extrahepatic neoplasms seen in children and young adults. Finally, FISH testing can be used as a diagnostic tool to confirm the diagnosis of FL-HCC, in the appropriate clinical setting.

Novel protein kinase cAMP-Activated Catalytic Subunit Alpha (PRKACA) inhibitor shows anti-tumor activity in a fibrolamellar hepatocellular carcinoma model.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is known as a highly aggressive liver cancer that typically affects young adults without virus infection. Since this type of cancer does not respond to chemotherapy, surgery is the only known effective therapeutic option. Most FL-HCC patients express the fusion gene DNAJB1-PRKACA, which has been recognized as the signature of FL-HCC. It has also been reported that PRKACA kinase activity is essential for its oncogenic activity, suggesting that PRKACA kinase inhibition could be considered as an useful therapeutic target. In this study, we established an evaluation system for PRKACA kinase inhibitors and synthesized DS89002333, a novel PRKACA inhibitor. DS89002333 showed potent PRKACA inhibitory activity and inhibited fusion protein-dependent cell growth both in vitro and in vivo. Furthermore, this compound showed anti-tumor activity in an FL-HCC patient-derived xenograft model expressing the DNAJB1-PRKACA fusion gene. Our data suggest that DS89002333 could be considered as a potential therapeutic agent for FL-HCC.

Adipocyte HIF2α functions as a thermostat via PKA Cα regulation in beige adipocytes.

Thermogenic adipocytes generate heat to maintain body temperature against hypothermia in response to cold. Although tight regulation of thermogenesis is required to prevent energy sources depletion, the molecular details that tune thermogenesis are not thoroughly understood. Here, we demonstrate that adipocyte hypoxia-inducible factor α (HIFα) plays a key role in calibrating thermogenic function upon cold and re-warming. In beige adipocytes, HIFα attenuates protein kinase A (PKA) activity, leading to suppression of thermogenic activity. Mechanistically, HIF2α suppresses PKA activity by inducing miR-3085-3p expression to downregulate PKA catalytic subunit α (PKA Cα). Ablation of adipocyte HIF2α stimulates retention of beige adipocytes, accompanied by increased PKA Cα during re-warming after cold stimuli. Moreover, administration of miR-3085-3p promotes beige-to-white transition via downregulation of PKA Cα and mitochondrial abundance in adipocyte HIF2α deficient mice. Collectively, these findings suggest that HIF2α-dependent PKA regulation plays an important role as a thermostat through dynamic remodeling of beige adipocytes.

DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling.

Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has a poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors; however, its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.

Bupleurum chinense DC improves CUMS-induced depressive symptoms in rats through upregulation of the cAMP/PKA/CREB signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Bupleurum chinense DC. (B. chinense) is the dried root of B. chinense, belonging to the Umbelliferae family. B. chinense has been reported since ancient times for its effect of soothing the liver and relieving depression. Additionally, its important role in treating depression, depressed mood disorders and anti-inflammation has been proven in previous studies. However, its specific mechanism of action remains unknown. AIM OF THE STUDY: The key targets and metabolites of the antidepressant effect of B. chinense were investigated based on the cAMP signalling pathway. The study examined the mechanism for the antidepressant effect of B. chinense by target prediction, analysis of related metabolites and potential metabolic pathways. MATERIALS AND METHODS: A network pharmacology approach was used to predict the antidepressant targets and pathways of B. chinense. A depression rat model was established through the CUMS (chronic unpredictable mild stress) procedure. The depression model was assessed by body weight, sugar-water preference, water maze and enzyme-linked immunosorbent assay (ELISA) indicators (5hydroxytryptamine, etc.). The key metabolic pathways were screened by correlations between metabolites and key targets. Finally, a quantitative analysis of key targets and metabolites was experimentally validated. RESULTS: B. chinense significantly ameliorated the reduction in body weight, sugar-water preference rate and cognitive performance in the water maze experiment in rats with depression induced by CUMS. ELISA, Western blotting (WB) and reverse transcription-polymerase chain reaction (RT-PCR) assays showed that B. chinense significantly improves the expression of protein kinase cyclic adenylic acid (cAMP)-activated catalytic subunit alpha (PRKACA), cAMP-response element-binding protein (CREB) and cAMP activation in the rat brain induced by CUMS. According to metabolic pathway analysis, B. chinense shows an antidepressant effect primarily by regulating the cAMP metabolic pathway. CONCLUSION: B. chinense upregulated PRKACA and CREB expression and the level of the key metabolite cAMP in the cAMP/PKA/CREB pathway while reducing the inflammatory response to depression treatment. These new findings support future research on the antidepressant effects of B. chinense.

Decabromodiphenyl ether-induced PRKACA hypermethylation contributed to glycolipid metabolism disorder via regulating PKA/AMPK pathway in rat and L-02 cells.

BDE-209 is the most prevalent congener of polybrominated diphenyl ethers and has high bioaccumulation in humans and animals. BDE-209 has been reported to disrupt glycolipid metabolism, but the mechanisms are still unclear. In this study, we found that BDE-209 induced liver tissue injury and hepatotoxicity, increased the glucose and total cholesterol levels in the serum of rats, and increased glucose and triglyceride levels in L-02 cells. BDE-209 exposure changed the PKA, p-PKA, AMPK, p-AMPK, ACC, and FAS expression in rats' liver and L-02 cells. Moreover, BDE-209 induced PRKACA-1 hypermethylation in L-02 cells. AMPK activator (AICAR) inhibited the changes of p-AMPK, ACC, and FAS expression and elevation of glucose and triglyceride levels induced by BDE-209. DNA methylation inhibitor (5-Aza-CdR) reversed BDE-209 induced alters of PKA/AMPK/ACC/FAS signaling pathway. These results demonstrated that BDE-209 could disrupt the glycolipid metabolism by causing PRKACA-1 hypermethylation to regulate the PKA/AMPK signaling pathway in hepatocytes.

Targeted Mutational Analysis of Cortisol-Producing Adenomas.

CONTEXT: Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPAs). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1. OBJECTIVE: This work aims to expand our understanding of the prevalence of somatic mutations in CPAs from patients with overt Cushing syndrome (OCS) and "subclinical" mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)‒guided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue. METHODS: We analyzed FFPE adrenal tissue from 77 patients (n = 12 men, 65 women) with either OCS (n = 32) or MACE (n = 45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPAs (32 OCS CPAs and 46 MACE CPAs). Genomic DNA was isolated from the FFPE CPAs and subjected to targeted amplicon sequencing for identification of somatic mutations. RESULTS: Somatic mutations were identified in 71.8% (56/78) of the CPAs. While PRKACA was the most frequently mutated gene in OCS CPAs (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE CPAs. Most GNAS mutations were observed in MACE CPAs (5/7, 71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored 2 adjacent tumors within the same adrenal gland - one patient had 2 CPAs, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase). CONCLUSION: A comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPAs. OCS CPAs demonstrated a distinct mutation profile compared to MACE CPAs.