To Explore the Inhibitory Mechanism of Quercetin in Thyroid Papillary Carcinoma through Network Pharmacology and Experiments.

Quercetin, a flavonoid with anti-inflammatory and anticancer properties, is expected to be an innovative anticancer therapeutic agent for papillary thyroid carcinoma (PTC). However, the downstream signaling pathways that mediate quercetin-dependent anticancer properties remain to be deciphered. Herein, potential targets of quercetin were screened with several bioinformatic avenues including PharmMapper, Gene Expression Omnibus (GEO) database, protein-protein interaction (PPI) network, and molecular docking. Besides, western blot, CCK-8 transwell analysis of migration and invasion, flow cytometric analysis, and colony formation assays were performed to investigate the underlying mechanism. We found four core nodes (MMP9, JUN, SPP1, and HMOX1) by constructing a PPI network with 23 common targets. Through functional enrichment analysis, we confirmed that the above four target genes are enriched in the TNF, PI3K-AKT, and NF-κB signaling pathways, which are involved in the inflammatory microenvironment and inhibit the development and progression of tumors. Furthermore, molecular docking results demonstrated that quercetin shows strong binding efficiency with the proteins encoded by these 4 key proteins. Finally, quercetin displayed strong antitumor efficacy in PTC cell lines. In this research, we demonstrated the application of network pharmacology in evaluating the mechanisms of action and molecular targets of quercetin, which regulates a variety of proteins and signaling pathways in PTC. These data might explain the mechanism underlying the anticancer effects of quercetin in PTC.

Targeting SHP2 sensitizes differentiated thyroid carcinoma to the MEK inhibitor.

Pharmacologic targeting of components of the MAPK/ERK pathway in differentiated thyroid carcinoma (DTC) is often limited due to the development of adaptive resistance. However, the detailed mechanism of MEK inhibitor (MEKi) resistance is not fully understood. Here, MEKi-resistant models were constructed successfully, in which multiple receptor tyrosine kinases (RTKs) signaling pathways and Src-homology 2 domain-containing phosphatase 2 (SHP2) were activated in MEKi-resistant cells. Given the physiological role of SHP2 as the downstream target of many RTKs, we first found blockade of SHP2 enhanced the sensitivity to MEKi in constructed MEKi-resistant models. Interestingly, we also found that compared with MEKi treatment alone, MEKi in combination with an SHP2 inhibitor markedly suppressed the reactivation of the MEK/ERK pathway; thus, the addition of the SHP2 inhibitor significantly improved the antitumor effects of MEKi. The synergistic suppression of DTC upon treatment with both inhibitors was further confirmed in xenograft models and transgenic models. Thus, our data suggest that RTKs activation leads to reactivation of the MAPK pathway and resistance to MEKi in DTC, which is reversed by SHP2 blockade. As a novel active inhibitor of SHP2, SHP099 in combination with MEKi is a promising therapeutic approach for advanced DTC and MEKi-resistant one.

Pharmacological inhibition of Ref-1 enhances the therapeutic sensitivity of papillary thyroid carcinoma to vemurafenib.

The use of the BRAF inhibitor vemurafenib exhibits drug resistance in the treatment of thyroid cancer (TC), and finding more effective multitarget combination therapies may be an important solution. In the present study, we found strong correlations between Ref-1 high expression and BRAF mutation, lymph node metastasis, and TNM stage. The oxidative stress environment induced by structural activation of BRAF upregulates the expression of Ref-1, which caused intrinsic resistance of PTC to vemurafenib. Combination inhibition of the Ref-1 redox function and BRAF could enhance the antitumor effects of vemurafenib, which was achieved by blocking the action of Ref-1 on BRAF proteins. Furthermore, combination treatment could cause an overload of autophagic flux via excessive AMPK protein activation, causing cell senescence and cell death in vitro. And combined administration of Ref-1 and vemurafenib in vivo suppressed PTC cell growth and metastasis in a cell-based lung metastatic tumor model and xenogeneic subcutaneous tumor model. Collectively, our study provides evidence that Ref-1 upregulation via constitutive activation of BRAF in PTC contributes to intrinsic resistance to vemurafenib. Combined treatment with a Ref-1 redox inhibitor and a BRAF inhibitor could make PTC more sensitive to vemurafenib and enhance the antitumor effects of vemurafenib by further inhibiting the MAPK pathway and activating the excessive autophagy and related senescence process.

Reduced MHC class II expression in medullary thyroid cancer identifies patients with poor prognosis.

Increasing body of recent studies determining the expression of tumor-specific major histocompatibility complex (MHC) class II protein supports its potential role in several malignancies, but little is known in human medullary thyroid cancer (MTC). Here, we report the expression of MHC-II and its clinicopathologic and prognostic relevance in MTC patients. Immunohistochemistry staining revealed a significant reduction in tumor cell-specific MHC-II expression in a higher AJCC stage and its poor prognostic correlation with human MTC development. Further statistical analysis identified the low MHC-II expression as a significant and independent risk factor for MTC recurrence and patient survival. Moreover, in vitro studies showed that the MHC-II expression was remarkably increased by RET inhibitors, which were prescribed to treat advanced MTC. Similarly, inhibitors blocking the MAPK/ERK and AKT/mTOR pathways also augmented MHC-II expression, suggesting their implications in RET-MHC-II signaling axis. Importantly, in vitro assays manifested enhanced peripheral blood leukocytes-mediated cytotoxicity in MTC cells treated with RET inhibitors, which were partially alleviated by HLA knock-down. Together, our study demonstrates that low MHC-II expression levels may serve as a prognostic biomarker for aggressive diseases in MTC patients and indicates that RET activation may promote MTC immune escape through downregulating MHC-II expression.

The prognostic landscape of tumor-infiltrating immune cells in lung squamous cell carcinoma.

BACKGROUND: Exploring novel biomarkers and developing effective therapeutic strategies can improve the prognosis of lung squamous cell carcinoma (LUSC) in the future. The prognostic value of tumor-infiltrating immune cells (TICs) in solid tumors has been extensively studied. However, the landscape of TICs involved in the prognosis of non-small cell lung cancer (NSCLC), especially in LUSC, remains unclear and should be systematically investigated. METHODS: This retrospective study analyzed the immune-related transcriptional profiles of 490 LUSC patients from The Cancer Genome Atlas (TCGA) cohort. Using the CIBERSORT method, TICs were evaluated and examined for their association with overall survival (OS) in LUSC. RESULTS: Out of the 27 TICs, 14 were correlated with prognosis in LUSC. A novel prognostic model characterized by fewer memory B cells and more central memory CD8 T cells, regulatory T cells (Tregs), and plasmacytoid dendritic cell (pDC) infiltration predicted poor OS in LUSC with high accuracy. The 1-, 3-, and 5-year areas under the curve (AUC) were 0.95, 0.98, and 0.96, respectively, in the training cohort. This finding was further validated in the validation cohort, where the 1-, 3-, and 5-year AUCs were 0.95, 0.98, and 0.95, respectively. CONCLUSIONS: These findings may provide more effective prognostic biomarkers and potential therapeutic targets for the immunotherapy of LUSC.

The mechanisms of celastrol in treating papillary thyroid carcinoma based on network pharmacology and experiment verification.

BACKGROUND: Celastrol, a triterpene present in the traditional Chinese medicine (TCM) Triptergium wilfordii, has been demonstrated to have remarkable anticancer activity. However, its specific mechanism on papillary thyroid carcinoma (PTC) remains to be elucidated. METHODS: Potential targets of celastrol were screened from public databases. Through the Gene Expression Omnibus (GEO) online database, we obtained the bioinformatics analysis profile of PTC, GSE33630, and analyzed the differentially expressed genes (DEGs). Then, a protein-protein interaction (PPI) network was constructed by utilizing the STRING database. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted. Finally, drug interactions between hub genes and celastrol were verified by molecular docking. RESULTS: Four core nodes (MMP9, JUN, ICAM1, and VCAM1) were discerned via constructing a PPI network of 47 common targets. Through functional enrichment analysis, it was confirmed that the above target genes were basically enriched in the interleukin-17 (IL-17), nuclear factor kappa-B (NF-κB), and tumor necrosis factor (TNF) signaling pathways, which are involved in the inflammatory microenvironment to inhibit the development and progression of tumors. Molecular docking results demonstrated that celastrol has a strong binding efficiency with the 4 key proteins. CONCLUSIONS: In this research, it was demonstrated that celastrol can regulate a variety of proteins and signaling pathways against PTC, providing a theoretical basis for future clinical applications.

Immune gene signature delineates a subclass of thyroid cancer with unfavorable clinical outcomes.

Thyroid cancer (THCA) is a heterogeneous disease with multiple clinical outcomes Immune cells regulate its progression. Three immunomolecular subtypes (C1, C2, C3) were identified in gene expression data sets from TCGA and GEO databases. Among them, subtype C3 had highest frequency of BRAF mutations, lowest frequency of RAS mutations, highest mutation load and shorter progression-free survival. Functional enrichment analysis for the genes revealed that C1 was up-regulated in the ERK cascade pathway, C2 was up-regulated in cell migration and proliferation pathways, while C3 was enriched in body fluid, protein regulation and response to steroid hormones functions. Notably, the three molecular subtypes exhibit differences in immune microenvironments as shown by timer database and analysis of immune expression signatures. The abundance of B_cell, CD4_Tcell, Neutrophil, Macrophage and Dendritic cells in C2 subtype were lower than in C1 and C3 subtypes Leukocyte fraction, proliferation macrophage regulation, lymphocyte infiltration, IFN gamma response and TGF beta response scores were significantly higher in C3 compared with C1 and C2 subtypes. Unlike C3 subtype, it was observed that C1 and C2 subtypes were significantly negatively correlated with most immune checkpoint genes in two different cohorts. The characteristic genes were differentially expressed between cancer cells, adjacent tissues, and metastatic tissues in different cohorts. In summary, THCA can be subclassified into three molecular subtypes with distinct histological types, genetic and transcriptional phenotypes, all of which have potential clinical implications.

microRNA­96 regulates the proliferation of nucleus pulposus cells by targeting ARID2/AKT signaling.

The aberrant proliferation of nucleus pulposus (NP) cells has been reported to be implicated in the pathogenesis of intervertebral disc degeneration (IDD). Previous studies have demonstrated that microRNAs (miRNAs), which are a group of small noncoding RNAs, are critical regulators of cell proliferation in various pathologies. However, the role of miRNA­96 (miR­96) in the proliferation of NP cells remains to be determined. In the present study, reverse transcription­quantitative polymerase chain reaction was used to investigate the expression of miR­96 in NP tissues from patients with IDD and healthy tissues from patients with traumatic lumbar fracture as the control. A dual­luciferase reporter assay was used to investigate whether AT­rich interaction domain 2 (ARID2) may be a direct target gene for miR­96. Furthermore, isolated NP cells from patients with IDD were transfected with miR­96 mimics and ARID2­targeting small interfering RNAs; cell proliferation, and the protein expression of Akt, phosphorylated Akt and ARID2 were examined, whereas the effects of an Akt inhibitor on NP cell proliferation were also evaluated. The present results demonstrated that miR­96 expression was significantly upregulated in IDD samples, and the level of miR­96 expression was positively associated with disc degeneration grade, which was evaluated by a modified Pfirrmann grading system. In addition, the current study identified ARID2 as a direct gene target of miR­96. Furthermore, it was demonstrated that ARID2 mRNA expression was inversely correlated with the expression of miR­96 in NP tissues. In addition, miR­96 overexpression promoted NP cell proliferation and induced Akt phosphorylation, which led to increased cyclin D1 translation. Notably, overexpression of ARID2 or treatment with an Akt inhibitor decreased the effect of miR­96 on NP cell proliferation. In conclusion, the results of the present study indicate that miR­96 may promote the proliferation of human degenerated NP cells by targeting ARID2 via activation of the Akt pathway, and potentially serves as a therapeutic target for IDD.

Utility of Fluorescence In Situ Hybridization (FISH) to Sub-Classify Low-Grade Urothelial Carcinoma for Prognostication.

BACKGROUND Fluorescence in situ hybridization (FISH) is used widely to detect cancer levels, but its value in urothelial carcinoma remains unclear. The aim of this study was to use FISH to examine the urine specimens of low-grade urothelial carcinoma (UC) patients to determine the possibility of sub-classifying the prognosis of UC. MATERIAL AND METHODS We diagnosed 107 patients with low-grade UC using a UroVysion kit to detect chromosomes 3, 7, 17, and P16 in the urine. An average 46.6-month follow-up completed in January 2016 combined with the clinical follow-up data were evaluated with Spearman's correlation analysis to analyze the aberration of chromosomes in relation to the prognostication. Univariate and multivariate analysis using the Mantel-Cox log-rank test for overall, cancer-specific, and disease-free survival were used to determine the prognostic significance of CSP7/CSP17 and CSP3/GLPp16. RESULTS In the 107 samples, 84 showed positive reaction in the FISH test. Furthermore, CSP7/CSP17 was found to be significantly related with age, tumor size, T stage, and tumor numbers, but not in CSP3/GLPp16. In addition, Kaplan-Meier analysis and Cox proportional hazards regression revealed a significant negative correlation between CSP7/CSP17 and survival, while CSP3/GLPp16 showed no significantly differences. CONCLUSIONS CSP7/CSP17 positivity on FISH test appears to play a critical role in low-grade UC and may be considered as a high-risk and prognosis factor.