The ERK inhibitor GDC-0994 selectively inhibits growth of BRAF mutant cancer cells.

BRAF or RAS mutation-induced aberrant activation of the mitogen-activated protein kinase (MAPK) pathway is frequently observed in human cancers. As the key downstream node of MAPK pathway, ERK1/2 is as an important therapeutic target. GDC-0994 (ravoxertinib), an orally bioavailable, highly selective small-molecule inhibitor of ERK1/2, showed acceptable safety and pharmacodynamic profile in a recent phase I clinical trial. In this study, we investigated dependence of the anti-tumor effect of ERK inhibitor GDC-0994 on genetic alterations in the MAPK pathway. The results showed that GDC-0994 sharply inhibited cell proliferation and colony formation and induced remarkable G1 phase cell-cycle arrest in cancer cells harboring BRAF mutation but had little effect on cell behaviors in most RAS mutant or wild-type cell lines. The expression of a large number of genes, particularly the genes in the cell cycle pathway, were significantly changed after GDC-0994 treatment in BRAF mutant cells, while no remarkable expression change of such genes was observed in wild-type cells. Moreover, GDC-0994 selectively inhibited tumor growth in a BRAF mutant xenograft mice model. Our findings demonstrate a BRAF mutation-dependent anti-tumor effect of GDC-0994 and provide a rational strategy for patient selection for ERK1/2 inhibitor treatment.

Antitumor activity of extracellular signal-regulated kinases 1/2 inhibitor BVD-523 (ulixertinib) on thyroid cancer cells.

OBJECTIVE: This study aimed to investigate BVD-523 (ulixertinib), an adenosine triphosphate (ATP)-dependent extracellular signal-regulated kinases 1/2 inhibitor, for its antitumor potential in thyroid cancer. MATERIALS AND METHODS: Ten thyroid cancer cell lines known to carry mitogen-activated protein kinase (MAPK)-activated mutations, including v-Raf murine sarcoma viral oncogene homolog B (BRAF) and rat sarcoma virus (RAS) mutations, were examined. Cells were exposed to a 10-fold concentration gradient ranging from 0 to 3000 nM for 5 days. The half-inhibitory concentration was determined using the Cell Counting Kit-8 assay. Following BVD-523 treatment, cell cycle analysis was conducted using flow cytometry. In addition, the impact of BVD-523 on extracellular signal-regulated kinase (ERK)- dependent ribosomal S6 kinase (RSK) activation and the expression of cell cycle markers were assessed through western blot analysis. RESULTS: BVD-523 significantly inhibited thyroid cancer cell proliferation and induced G1/S cell cycle arrest dose-dependently. Notably, cell lines carrying MAPK mutations, especially those with the BRAF V600E mutation, exhibited heightened sensitivity to BVD-523's antitumor effects. Furthermore, BVD-523 suppressed cyclin D1 and phosphorylated retinoblastoma protein expression, and it robustly increased p27 levels in an RSK-independent manner. CONCLUSION: This study reveals the potent antitumor activity of BVD-523 against thyroid cancer cells bearing MAPK-activating mutations, offering promise for treating aggressive forms of thyroid cancer.

Dihydroartemisinin (DHA) inhibits myofibroblast differentiation through inducing ferroptosis mediated by ferritinophagy.

Idiopathic pulmonary fibrosis (IPF) is caused by persistent micro-injuries and aberrant repair processes. Myofibroblast differentiation in lung is a key event for abnormal repair. Dihydroartemisinin(DHA), a well-known anti-malarial drug, have been shown to alleviate pulmonary fibrosis, but its mechanism is not clear. Ferroptosis is involved in the pathgenesis of many diseases, including IPF. Ferritinophagy is a form of cellular autophagy which regulates intracellular iron homeostasis. The function of DHA on myofibroblasts differentiation of pulmonary and whether related with ferroptosis and ferritinophagy are unknown now. Using human fetal lung fibroblast 1(HFL1) cell line and the qRT-PCR, immunofluorescent and Western blotting techniques, we found that after TGF-ß1 treatment, the levels of ɑ-SMA expression and ROS increased; the mRNA and protein levels of FTH1 and NCOA4, the content of Fe2+ and 4-HNE increased significantly at 6h, then gradually reduced with time. After DHA treatment, FHL1 cells appeared ferroptosis; the levels of α-SMA mRNA and protein reduced and the levels of ROS and 4-HNE increased; the Fe2+ levels decreased sharply at 6h, then increased with time, and were higher than normal since 24h; the mRNA and protein levels of FTH1 and NCOA4 decreased, exhibited a downward trend. These results show that Fe2+, ROS and lipid peroxidation are involved in and ferritinophagy is inhibited during fibroblast-to-myofibroblast differentiation; The depletion of Fe2+ at early stage induced by DHA treatment triggers the ferritinophagy in HFL1 cells, leading to degradation of FTH1 and NCOA4 and following increase of Fe2+ levels. DHA may inhibit the fibroblast-to-myofibroblast differentiation through inducing ferroptosis mediated by ferritinophagy.

TERT promoter methylation is associated with high expression of TERT and poor prognosis in papillary thyroid cancer.

The telomerase reverse transcriptase (TERT) is overexpressed and associated with poor prognosis in papillary thyroid cancer (PTC), the most common subtype of thyroid cancer. The overexpression of TERT in PTC was partially attributed to transcriptional activation by two hotspot mutations in the core promoter region of this gene. As one of the major epigenetic mechanisms of gene expression regulation, DNA methylation has been proved to regulate several tumor-related genes in PTC. However, the association of TERT promoter DNA methylation with TERT expression and PTC progression is still unclear. By treating PTC cell lines with demethylating agent decitabine, we found that the TERT promoter methylation and the genes' expression were remarkably decreased. Consistently, PTC patients with TERT hypermethylation had significantly higher TERT expression than patients with TERT hypomethylation. Moreover, TERT hypermethylated patients showed significant higher rates of poor clinical outcomes than patients with TERT hypomethylation. Results from the cox regression analysis showed that the hazard ratios (HRs) of TERT hypermethylation for overall survival, disease-specific survival, disease-free interval (DFI) and progression-free interval (PFI) were 4.81 (95% CI, 1.61-14.41), 8.28 (95% CI, 2.14-32.13), 3.56 (95% CI, 1.24-10.17) and 3.32 (95% CI, 1.64-6.71), respectively. The HRs for DFI and PFI remained significant after adjustment for clinical risk factors. These data suggest that promoter DNA methylation upregulates TERT expression and associates with poor clinical outcomes of PTC, thus holds the potential to be a valuable prognostic marker for PTC risk stratification.

Risk stratification by combining common genetic mutations and TERT promoter methylation in papillary thyroid cancer.

PURPOSE: Risk stratification based on somatic mutations in TERT promoter and BRAF/RAS has been well established for papillary thyroid cancer (PTC), and there is emerging evidence showed that TERT promoter methylation was frequently observed in thyroid cancer patients with adverse features. This study was aimed to comprehensive explore the prognostic value of BRAF/RAS mutations, TERT promoter mutations, and TERT promoter methylation in PTC. METHODS: The relationships of BRAF/RAS mutations, TERT promoter mutations, and TERT promoter methylation with clinical characteristics and outcomes of PTC were analyzed in 382 patients with PTC. RESULTS: TERT promoter mutation and hypermethylation were collectively observed in 52 (13.6%) samples and associated with BRAF/RAS mutation, aggressive clinical characteristics, and poor clinical outcomes of PTC. Coexistence of BRAF/RAS and TERT alterations was found in 45 of 382 (11.8%) PTC patients and strongly associated with old patient age, extrathyroidal extension, advanced pathologic T stage and metastasis. Importantly, patients with both BRAF/RAS and TERT alterations had higher rates of tumor recurrence (13.6% vs 1.5%, P = 0.042) and disease progression (24.4% vs 3.3%, P < 0.001) than patients without any alterations, and cox regression analysis revealed that the coexistence of BRAF/RAS and TERT alterations, but not BRAF/RAS or TERT alterations alone, increased the risk of progression-free interval with an adjusted HR of 10.35 (95% CI: 1.79-59.81, P = 0.009). CONCLUSIONS: This study suggested that comprehensively analysis of BRAF/RAS mutations, TERT promoter mutation and methylation is an effective strategy to identify high-risk patients with PTC.

Methylation-Mediated Silencing of ATF3 Promotes Thyroid Cancer Progression by Regulating Prognostic Genes in the MAPK and PI3K/AKT Pathways.

Background: Aberrant expression of oncogenes and/or tumor suppressor genes (TSGs) drives the tumorigenesis and development of thyroid cancer. We investigated the expression and function of a member of the activating transcription factor (ATF)/cAMP-responsive element-binding protein (CREB) transcription factor (TF) family, ATF3, in thyroid cancer. Methods: Data from 80 patients with papillary thyroid cancer (PTC) in the First Affiliated Hospital of Sun Yat-sen University and 510 PTC samples in The Cancer Genome Atlas thyroid cancer database were utilized for gene expression and prognosis analyses. The survival data were analyzed by Kaplan-Meier curves and Cox regression with adjustment for age, sex, multilocality, extrathyroidal extension, lymph metastases, and history of neoadjuvant treatment. DNA methylation was analyzed by methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing PCR. TFs binding to ATF3 promoter were identified by DNA pull-down combined with mass spectrum assay, and confirmed by quantitative PCR (qPCR), luciferase reporter assay, and chromatin immunoprecipitation (ChIP)-qPCR. We conducted functional assays in vitro and in a xenograft mouse model to evaluate the function of ATF3 in thyroid cancer. Integrated analyses based on RNA sequencing, ChIP-seq, and CUT&Tag assays were performed to explore the mechanisms underlying the function of ATF3. Results: ATF3 was significantly downregulated in PTC and patients with low ATF3 expression had reduced progression-free survival (adjusted hazard ratio = 0.50 [CI 0.26-0.98], p = 0.043). DNA hypermethylation in ATF3 promoter disrupted the binding of SP1 and MYC-MAX, leading to inactivation of the gene. ATF3 functioned as a TSG by inhibiting the proliferation and mobility of thyroid cancer cells. And ATF3 regulated the expression of a number of genes by binding to the regulatory elements of them, particularly for genes in MAPK and PI3K/AKT pathways. Among these target genes, filamin C was positively regulated by ATF3 and associated with a more favorable thyroid cancer prognosis, while dual specificity phosphatase 10, fibronectin-1, tenascin C, and CREB5 were negatively regulated by ATF3 and associated with a poorer prognosis. Conclusions: We observed that the promoter DNA hypermethylation decreased the expression of ATF3, which in turn promoted the progression of thyroid cancer, at least partially, by directly regulating prognosis-related genes in the MAPK and PI3K/AKT pathways.

Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer.

BACKGROUND: Thyroid cancer is the most frequent malignancy of the endocrine system, of which papillary thyroid cancer (PTC) is the predominant form with a rapid increasing incidence worldwide. Rearranged during transfection (RET) fusions are common genetic drivers of PTC and the potent RET inhibitor selpercatinib has been recently approved for treating advanced or metastatic RET fusion-positive thyroid cancer. In this study we aimed to develop a droplet digital PCR (ddPCR) system to accurately detect RET fusion in PTC samples. METHODS: The frequency and distribution of RET fusions in PTC were analyzed using genomic data of 402 PTC patients in The Cancer Genome Atlas (TCGA) database. To establish the ddPCR system for detecting CCDC6::RET fusion, a plasmid containing CCDC6::RET infusion fragment was constructed as standard template, the annealing temperature and concentrations of primers and probe were optimized. The analytical performance of ddPCR and quantitative reverse transcription PCR (qRT-PCR) were assessed in standard templates and tissue samples from 112 PTC patients. Sanger sequencing was performed in all the RET fusion-positive samples identified by ddPCR. RESULTS: RET fusions were observed in 25 (6.2%) of the 402 TCGA samples, and 15 (60%) of the RET fusion-positive patients had the CCDC6::RET fusion. Compared with qRT-PCR, the ddPCR method showed a lower limit of detection (128.0 and 430.7 copies/reaction for ddPCR and qRT-PCR, respectively). When applying the two methods to 112 tissue samples of PTC, eleven (9.8%) CCDC6::RET fusion-positive samples were detected by qRT-PCR, while ddPCR identified 4 additional positive samples (15/112, 13.4%). All the CCDC6::RET fusion-positive cases identified by ddPCR were confirmed by Sanger sequencing except for one case with 0.14 copies/uL of the fusion. CONCLUSION: The accurate and sensitive ddPCR method reported here is powerful to detection CCDC6::RET fusion in PTC samples, application of this method would benefit more RET fusion-positive patients in the clinic.

STRA6 Promotes Thyroid Carcinoma Progression via Activation of the ILK/AKT/mTOR Axis in Cells and Female Nude Mice.

BACKGROUND: Metastasis has emerged to be an important cause for poor prognosis of thyroid carcinoma (TC) and its molecular mechanisms are not fully understood. STRA6 is a multifunctional membrane protein widely expressed in embryonic and adult tissues. The function and mechanism of STRA6 in TC remain elusive. OBJECTIVE: We aimed to explore the role of STRA6 in TC progression and provide a therapeutic target for TC. METHODS: The expression and clinicopathological relevance of STRA6 were explored in TC. Stable STRA6-knockdown TC cells were established and used to determine the biological function of STRA6 in vitro and in vivo. RNA sequencing and co-immunoprecipitation were performed to unveil the molecular mechanism of STRA6 in TC progression. The potential of STRA6 as a therapeutic target was evaluated by lipid nanoparticles (LNPs) containing siRNA. RESULTS: STRA6 was upregulated in TC and correlated with aggressive clinicopathological features, including extrathyroidal extension and lymph node metastasis, which contributed to the poor prognosis of TC. STRA6 facilitated TC progression by enhancing proliferation and metastasis in vitro and in vivo. Mechanistically, STRA6 could interact with integrin-linked kinase (ILK) and subsequently activate the protein kinase B/mechanistic target of rapamycin (AKT/mTOR) signaling pathway. We further unveiled that STRA6 reprogrammed lipid metabolism through SREBP1, which was crucial for the metastasis of TC. Moreover, STRA6 siRNA delivered by LNPs significantly inhibited cell growth in xenograft tumor models. CONCLUSIONS: Our study demonstrates the critical roles of STRA6 contributing to TC progression via the ILK/AKT/mTOR axis, which may provide a novel prognostic marker as well as a promising therapeutic target for aggressive TC.

The ETS Inhibitor YK-4-279 Suppresses Thyroid Cancer Progression Independent of TERT Promoter Mutations.

Hotspot mutations in the core promoter region of the telomerase reverse transcriptase (TERT) gene have been well established to associate with aggressive clinical characteristics, radioiodine refractory, tumor recurrence, and mortality in thyroid cancer. Several E-twenty-six (ETS) transcription factors were reported to selectively bound to the mutant TERT promoter and activated TERT expression. In this study we aimed to investigate whether TERT promoter mutations confer sensitivity to ETS inhibitor YK-4-279 in thyroid cancer cells and whether this inhibitor could be served as a potential therapeutic agent for thyroid cancer. In vitro assays showed that YK-4-279 treatment sharply suppressed cell viability, colony formation, migration, and invasion, as well as induced cell cycle arrest and apoptosis in a panel of thyroid cancer cells. The cell viability after YK-4-279 treatment was similar between cell lines harboring mutant and wild-type TERT promoters. Furthermore, YK-4-279 treatment reduced both luciferase activity and mRNA expression of TERT independent of TERT promoter mutation status. Data from RNA-seq further revealed that YK-4-279 significantly affected biological processes including DNA replication and cell cycle. Reduced DNA helicase activity and decreased expression of several helicase genes were observed after YK-4-279 treatment. Moreover, YK-4-279 significantly inhibited tumor growth and induced apoptosis in a xenograft mice model. Thus, ETS inhibitor YK-4-279 suppressed TERT expression and conferred anti-tumor activity in a TERT promoter mutation-independent manner, and it could be a potential agent for the treatment of advanced thyroid cancers.

Selenite Induces Cell Cycle Arrest and Apoptosis via Reactive Oxygen Species-Dependent Inhibition of the AKT/mTOR Pathway in Thyroid Cancer.

Thyroid cancer is the most common endocrine malignancy, and its incidence has increased in the past decades. Selenium has been shown to have therapeutic effects against several tumors. However, its role in thyroid cancer and its underlying molecular mechanism remains to be explored. In the present study, we demonstrated that sodium selenite significantly decreased cell viability and induced G0/G1 cell cycle arrest and apoptosis in thyroid cancer cells in a dose-dependent manner. Transcriptomics revealed that sodium selenite induced intracellular reactive oxygen species (ROS) by promoting oxidative phosphorylation. Increased intracellular ROS levels inhibited the AKT/mTOR signaling pathway and upregulated EIF4EBP3. Intracellular ROS inhibition by N-acetylcysteine (NAC) ameliorated the cellular effects of sodium selenite. The in vitro findings were reproduced in xenograft thyroid tumor models. Our data demonstrated that sodium selenite exhibits strong anticancer effects against thyroid cancer cells, which involved ROS-mediated inhibition of the AKT/mTOR pathway. This suggests that sodium selenite may serve as a therapeutic option for advanced thyroid cancer.

Effects of exendin-4 on the intrarenal renin-angiotensin system and interstitial fibrosis in unilateral ureteral obstruction mice: Exendin-4 and unilateral ureteral obstruction.

OBJECTIVE: The objective of this article is to investigate the renoprotecive effects of exendin-4 in a mouse model of unilateral ureteral obstruction (UUO) and explore the putative mechanisms. METHODS: Male Balbc mice underwent sham operation or UUO surgery, and then received intraperitoneal injection of vehicle or exendin-4, respectively. After 14 days, mice were sacrificed and the left kidneys were collected and analyzed by histology, immunohistochemistry, Western blot, quantitative real-time reverse transcription polymerase chain reaction, radioimmunoassay and enzyme-linked immunosorbent assay. RESULTS: As compared to the sham group, mice that underwent UUO surgery developed more severe tubular injury and interstitial fibrosis, as well as higher expression of fibronectin (FN), collagen-1 (Col-1) and α-smooth muscle actin (α-SMA). Also, we observed higher expression of angiotensin-converting enzyme (ACE) while lower expression of angiotensin-converting enzyme 2 (ACE2), higher levels of intrarenal angiotensin II (Ang II) while lower levels of intrarenal angiotensin-(1-7), and higher expression of transforming growth factor ß1 (TGF-ß1) and phosphorylation of Smad3 (p-Smad3) in the obstructed kidneys. Impressively, these pathologic changes were significantly attenuated in the mice group of UUO treated with exendin-4. CONCLUSION: Our present study indicates for the first time that exendin-4 exerts renoprotective effects in an experimental model of UUO, partly through regulating the balance of the intrarenal renin-angiotensin system and then inhibiting the Ang II-mediated TGF-ß1/Smad3 signaling pathway.

Let-7d miRNA prevents TGF-ß1-induced EMT and renal fibrogenesis through regulation of HMGA2 expression.

TGF-ß1-induced epithelial to mesenchymal transition (EMT) process of tubular epithelial cells plays a leading role in the occurrence and progression of renal fibrosis as seen in diabetic nephropathy (DN). High mobility group AT-hook 2 (HMGA2) is considered to be involved in TGF-ß1-mediated EMT via multifactorial mechanisms. Specific microRNAs (miRNAs) are closely associated with EMT, and here we focused on let-7d miRNA as a regulator of HMGA2. This study aims to investigate the effects of HMGA2 on EMT process induced by TGF-ß1 using small interfering RNA (siRNA) technique in vitro, and further explore the potential role of let-7d miRNA during renal fibrosis in DN. We demonstrated that siRNA targeting HMGA2 was sufficient to inhibit TGF-ß1-induced EMT and fibrogenesis in rat kidney tubular epithelial cells (NRK52E). Furthermore, let-7d expression was significantly reduced by TGF-ß1 stimulation, we focused on let-7d and found that overexpression of let-7d down-regulated the expression of HMGA2 and in turn suppressed TGF-ß1-induced EMT and renal fibrogenesis. Inhibition of let-7d increased HMGA2 expression and enhanced the profibrogenic effects of TGF-ß1 on NRK-52E cells. Consistent with the above observations in vitro, let-7d expression was also decreased in the kidneys of unilateral ureter obstruction model, accompanied by the correspondingly increased expression of HMGA2 and fibrotic genes in this model. Collectively, HMGA2 and let-7d miRNA significantly impact on the progression of TGF-ß1-induced EMT and fibrogenesis both in vitro and in vivo, and they may represent novel targets for the prevention strategies of renal fibrosis in the context of DN.