Pharmacogenomic analysis in adrenocortical carcinoma reveals genetic features associated with mitotane sensitivity and potential therapeutics.

Background: Adrenocortical carcinoma (ACC) is an aggressive endocrine malignancy with limited therapeutic options. Treating advanced ACC with mitotane, the cornerstone therapy, remains challenging, thus underscoring the significance to predict mitotane response prior to treatment and seek other effective therapeutic strategies. Objective: We aimed to determine the efficacy of mitotane via an in vitro assay using patient-derived ACC cells (PDCs), identify molecular biomarkers associated with mitotane response and preliminarily explore potential agents for ACC. Methods: In vitro mitotane sensitivity testing was performed in 17 PDCs and high-throughput screening against 40 compounds was conducted in 8 PDCs. Genetic features were evaluated in 9 samples using exomic and transcriptomic sequencing. Results: PDCs exhibited variable sensitivity to mitotane treatment. The median cell viability inhibition rate was 48.4% (IQR: 39.3-59.3%) and -1.2% (IQR: -26.4-22.1%) in responders (n=8) and non-responders (n=9), respectively. Median IC50 and AUC were remarkably lower in responders (IC50: 53.4 µM vs 74.7 µM, P<0.0001; AUC: 158.0 vs 213.5, P<0.0001). Genomic analysis revealed CTNNB1 somatic alterations were only found in responders (3/5) while ZNRF3 alterations only in non-responders (3/4). Transcriptomic profiling found pathways associated with lipid metabolism were upregulated in responder tumors whilst CYP27A1 and ABCA1 expression were positively correlated to in vitro mitotane sensitivity. Furthermore, pharmacologic analysis identified that compounds including disulfiram, niclosamide and bortezomib exhibited efficacy against PDCs. Conclusion: ACC PDCs could be useful for testing drug response, drug repurposing and guiding personalized therapies. Our results suggested response to mitotane might be associated with the dependency on lipid metabolism. CYP27A1 and ABCA1 expression could be predictive markers for mitotane response, and disulfiram, niclosamide and bortezomib could be potential therapeutics, both warranting further investigation.

Comprehensive analysis of the m6A-related molecular patterns and diagnostic biomarkers in osteoporosis.

Background: N6-methyladenosine (m6A) modification is a critical epigenetic modification in eukaryotes and involves several biological processes and occurrences of diseases. However, the roles and regulatory mechanisms of m6A regulators in osteoporosis (OP) remain unclear. Thus, the purpose of this study is to explore the roles and mechanisms of m6A regulators in OP. Methods: The mRNA and microRNA (miRNA) expression profiles were respectively obtained from GSE56815, GSE7158, and GSE93883 datasets in Gene Expression Omnibus (GEO). The differential expression of 21 m6A regulators between high-bone mineral density (BMD) and low-BMD women was identified. Then, a consensus clustering of low-BMD women was performed based on differentially expressed (DE)-m6A regulators. The m6A-related differentially expressed genes (DEGs), the differentially expressed miRNAs (DE-miRNAs), and biological functions were investigated. Moreover, a weighted gene co-expression network analysis (WGCNA) was constructed to identify the OP-related hub modules, hub genes, and the functional pathways. Then, an m6A regulator-target-pathway network and the competing endogenous RNA (ceRNA) network in key modules were constructed. A least absolute shrinkage and selection operation (LASSO) Cox regression model and a Support Vector Machine-Recursive Feature Elimination (SVM-RFE) model were constructed to identify the candidate genes for OP prediction. The receiver operator characteristic (ROC) curves were used to validate the performances of predictive models and candidate genes. Results: A total of 10,520 DEGs, 13 DE-m6A regulators, and 506 DE-miRNAs between high-BMD and low-BMD women were identified. Two m6A-related subclusters with 13 DE-m6A regulators were classified for OP. There were 5,260 m6A-related DEGs identified between two m6A-related subclusters, the PI3K-Akt, MAPK, and immune-related pathways, and bone metabolism was mainly enriched in cluster 2. Cell cycle-related pathways, RNA methylation, and cell death-related pathways were significantly involved in cluster 1. Five modules were identified as key modules based on WGCNA, and an m6A regulator-target gene-pathway network and the ceRNA network were constructed in module brown. Moreover, three m6A regulators (FTO, YTHDF2, and CBLL1) were selected as the candidate genes for OP. Conclusion: M6A regulators play an important role in the occurrences and diagnosis of OP.

Network Pharmacology and Molecular Docking Elucidate the Pharmacological Mechanism of the OSTEOWONDER Capsule for Treating Osteoporosis.

Background: Osteoporosis (OP) is a serious and common bone metabolic disease with bone mass loss and bone microarchitectural deterioration. The OSTEOWONDER capsule is clinically used to treat OP. However, the potential regulatory mechanism of the OSTEOWONDER capsule in treatment of OP remains largely unknown. Methods: The bioactive compounds of herbs and their targets were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. The speculative targets of OP were screened out based on GeneCards, DisGeNET, and Online Mendelian Inheritance in Man (OMIM) databases. The gene modules and hub genes of OP were identified using a weighted gene co-expression network analysis (WGCNA). Then, an herb-compound-target network was constructed based on the above analyses. The biological function of targets was subsequently investigated, and a protein-protein interaction (PPI) network was constructed to identify hub targets of OP. Finally, molecular docking was performed to explore the interaction between compounds and targets. Results: A total of 148 compounds of eight herbs and the corresponding 273 targets were identified based on the TCMSP database. A total of 4,929 targets of OP were obtained based on GeneCards, DisGeNET, and OMIM databases. In addition, six gene modules and 4,235 hub genes of OP were screened out based on WGCNA. Generally, an herb-compound-target network, including eight herbs, 84 compounds, and 58 targets, was constructed to investigate the therapeutic mechanism of the OSTEOWONDER capsule for OP. The biofunction analysis indicated 58 targets mainly associated with the bone metabolism, stimulation response, and immune response. EGFR, HIF1A, MAPK8, IL6, and PPARG were identified as the hub therapeutic targets in OP. Moreover, the interaction between EGFR, HIF1A, MAPK8, IL6, PPARG, and the corresponding compounds (quercetin and nobiletin) was analyzed using molecular docking. Conclusion: Our finding discovered the possible therapeutic mechanisms of the OSTEOWONDER capsule and supplied the potential therapeutic targets for OP.

Caveolin-1 Is Essential for the Improvement of Insulin Sensitivity through AKT Activation during Glargine Treatment on Diabetic Mice.

Insulin treatment was confirmed to reduce insulin resistance, but the underlying mechanism remains unknown. Caveolin-1 (Cav-1) is a functional protein of the membrane lipid rafts, known as caveolae, and is widely expressed in mammalian adipose tissue. There is increasing evidence that show the involvement of Cav-1 in the AKT activation, which is responsible for insulin sensitivity. Our aim was to investigate the effect of Cav-1 depletion on insulin sensitivity and AKT activation in glargine-treated type 2 diabetic mice. Mice were exposed to a high-fat diet and subject to intraperitoneal injection of streptozotocin to induce diabetes. Next, glargine was administered to treat T2DM mice for 3 weeks (insulin group). The expression of Cav-1 was then silenced by injecting lentiviral-vectored short hairpin RNA (shRNA) through the tail vein of glargine-treated T2DM mice (CAV1-shRNA group), while scramble virus injection was used as a negative control (Ctrl-shRNA group). The results showed that glargine was able to upregulate the expression of PI3K and activate serine phosphorylation of AKT through the upregulation of Cav-1 expression in paraepididymal adipose tissue of the insulin group. However, glargine treatment could not activate AKT pathway in Cav-1 silenced diabetic mice. These results suggest that Cav-1 is essential for the activation of AKT and improving insulin sensitivity in type 2 diabetic mice during glargine treatment.

Caveolin-1 deficiency protects pancreatic ß cells against palmitate-induced dysfunction and apoptosis.

Lipotoxicity leads to insulin secretion deficiency, which is among the important causes for the onset of type 2 diabetes mellitus. Thus, the restoration of ß-cell mass and preservation of its endocrine function are long-sought goals in diabetes research. Previous studies have suggested that the membrane protein caveolin-1 (Cav-1) is implicated in ß-cell apoptosis and insulin secretion, however, the underlying mechanisms still remains unclear. Our objective is to explore whether Cav-1 depletion protects pancreatic ß cells from lipotoxicity and what are the underlying mechanisms. In this study, we found that Cav-1 silencing significantly promoted ß-cell proliferation, inhibited palmitate (PA)-induced pancreatic ß-cell apoptosis and enhanced insulin production and secretion. These effects were associated with enhanced activities of Akt and ERK1/2, which in turn downregulated the expression of cell cycle inhibitors (FOXO1, GSK3ß, P21, P27 and P53) and upregulated the expression of Cyclin D2 and Cyclin D3. Subsequent inhibition of PI3K/Akt and ERK/MAPK pathways abolished Cav-1 depletion induced ß-cell mass protection. Furthermore, under PA induced endoplasmic reticulum (ER) stress, Cav-1 silencing significantly reduced eIF2α phosphorylation and the expression of ER stress-responsive markers BiP and CHOP, which are among the known sensitizers of lipotoxicity. Our findings suggest Cav-1 as potential target molecule in T2DM treatment via the preservation of lipotoxicity-induced ß-cell mass reduction and the attenuation of insulin secretion dysfunction.

The effects of caveolin1 on ß cell proliferation.

Our study aims to access the influence of caveolin1 (CAV1) on ß cell expression profiles. We knocked down the expression of CAV1 in both NIT-1 cells and islets isolated from C57BL/6J mice using an RNA interference technique, which was realized by the transfer of an shRNA vector targeting CAV1 mRNA into NIT-1 cells or islets through latent virus infection. First, we identified the change in gene expression profiles in islets, in which the CAV1 expression level was down-regulated, as ascertained by mouse gene expression microarray, and the results showed that pathways related to ß cell proliferation and pancreatic secretion functions were significantly influenced. The results of MTT demonstrated that the knockdown of CAV1 expression in NIT-1 cells promoted proliferation. The protein array results showed that pro-apoptotic cytokines were down-regulated in the NIT-1 cell line with CAV1 knockdown. These findings suggest that CAV1 might be involved in apoptosis and proliferation regulation in ß cells, and therefore could be a potential target for the development of novel therapies for diabetes mellitus.