Bisphenol A at a human exposed level can promote epithelial-mesenchymal transition in papillary thyroid carcinoma harbouring BRAFV600E mutation

Bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical, alters the function of endocrine system and enhances the susceptibility to tumorigenesis in several hormone-dependent tumours as thyroid carcinoma. About 50% of papillary thyroid cancers (PTC), the most common type of thyroid malignancy, harbours the BRAF^V600E mutation. This study aimed to investigate a potential combined effect of BPA exposure and BRAF^V600E mutation on epithelial-mesenchymal transition (EMT) in PTC. Firstly, the level of BPA in plasma, the evaluation of BRAF^V600E mutation and the level of EMT-related proteins in PTC samples were individually determined. Additionally, the migration, invasion, colony formation capacity and the expression of EMT-related proteins after exposure to BPA were precisely analysed in vitro thyroid cells genetically modified by the introduction of BRAF^V600E mutation. Moreover, ERK-Cox2 signalling pathway was also introduced to explore the possible mechanism in PTC development. As expected, whether the clinical investigation or cultured thyroid cells demonstrated that BPA at a concentration compatible with human exposed levels (10^-7 M) synergized with the BRAF^V600E mutation promoted EMT via the activation of ERK-Cox2 signalling pathway. Our findings offer some evidence that BPA as an environmental risk factor can facilitate the progression of PTC harbouring BRAF^V600E mutation.     

Characterizing the three-dimensional organization of telomeres in papillary thyroid carcinoma cells

The relationship between the three-dimensional (3D) nuclear telomere architecture and specific genetic alterations in papillary thyroid carcinoma (PTC), in particular in cancer stem-like cells (CSLCs), has not yet been investigated. We isolated thyrospheres containing CSLCs from B-CPAP, K1, and TPC-1 PTC-derived cell lines, representative of tumors with different genetic backgrounds within the newly identified BRAF^V600E-like PTC subgroup, and used immortalized normal human thyrocytes (Nthy-ori 3.1) as control. We performed quantitative fluorescence in situ hybridization, 3D imaging, and 3D telomere analysis using TeloView software to examine telomere dysfunction in both parental and thyrosphere cells. Among the 3D telomere profile, a wide heterogeneity was observed, except for telomere intensity. Our findings indicate that CSLCs of each cell line had longer telomeres than parental cells, according to telomere intensity values, which correlate with telomere length. Indeed, the thyrosphere cells had lower numbers of lower-intensity telomeres (≤5,000 arbitrary fluorescent units, a.u.), compared with parental cancer cells, as well as parental control cells, (p < 0.0001). The B-CPAP thyrospheres showed a decreased number of higher intensity telomeres (>17,000 a.u.) than K1 and TPC-1 cells, as well as control cells (p < 0.0001). By selecting PTC-derived cell lines with different genetic backgrounds characteristic of BRAF^V600E-like PTC subgroups, we demonstrate that thyrosphere cells with BRAF^V600E and TP53 mutations show shorter telomeres than those harboring RET/PTC or BRAF^V600Eand wild-type TP53. Hence, our data reveal a trend towards a decrease in telomere shortening in CSLCs, representing the early cancer-promoting subpopulation, as opposed to parental cells representing the tumor bulk cells.     

Regulating the response to targeted MEK inhibition in melanoma

The limitations of revolutionary new mutation-specific inhibitors of BRAF^V600E include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAF^V600E by PLX4720 synergistically induces apoptosis across a spectrum of BRAF^V600E melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.     

Expression levels of HMGA2 and CD9 and its clinicopathological significances in the benign and malignant lesions of the gallbladder

Background

Papillary thyroid carcinoma (PTC) is one of the most frequent endocrine malignancies. In most cases, it often presents as multifocal tumor. It has been reported that multifocal tumors are associated with elevated risk of lymph node and distant metastases. Multifocality is also one of the factors predicting prognosis. Recent studies show that BRAF^V600E mutation occurs more frequently in aggressive PTC. The purpose of this study was to evaluate BRAF^V600E status and clinicopathological features in multiple and solitary PTC.

Methods

We performed a retrospective study to analyze 512 PTC cases who received surgery, including 376 solitary PTCs and 136 multiple PTCs.

Results

Multiple PTC is more related to lymph node metastasis and vascular invasion than solitary PTC. However, the distant metastasis rate and 10-year survival rate showed no difference between these two groups. BRAF^V600E mutation status was more frequent in multiple PTC patients with lymph node metastasis and late stage at diagnosis.

Conclusion

BRAF^V600E mutation is most commonly associated with extra-thyroidal extension and lymph node metastasis in PTC. Multiple PTC patients with young age, large tumors and BRAF^V600E mutation should be followed carefully. Our study provides useful information for PTC patients?? followup and treatment.     

Hypermethylated RASSF1 and SLC5A8 promoters alongside BRAFV600E mutation as biomarkers for papillary thyroid carcinoma

Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAF^V600E mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAF^V600E mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAF^V600E mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAF^V600E mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination.     

Associations of the BRAFV600E Mutation with Sonographic Features and Clinicopathologic Characteristics in a Large Population with Conventional Papillary Thyroid Carcinoma

Objective

To evaluate the association of the BRAF^V600E mutation with sonographic features and clinicopathologic characteristics in a large population with conventional papillary thyroid carcinoma (PTC).

Methods

We retrospectively reviewed the sonographic features, clinicopathologic characteristics, and presence of the BRAF^V600E mutation in 688 patients who underwent thyroidectomy for conventional PTC between January and July 2010 at a single institution. The incidence of the BRAF^V600E mutation was calculated. The sonographic features and clinicopathologic characteristics were compared between BRAF-positive and BRAF-negative patients. BRAF-positive patients were subdivided into those with papillary thyroid microcarcinoma (the PTMC group) and those with PTC larger than 10 mm (the PTC>10 mm group), and their sonographic features were compared.

Results

The BRAF^V600E mutation was detected in 69.2% of patients (476 of 688). Sonographic features were not significantly different between BRAF-positive and BRAF-negative PTC, nor between PTMC and PTC>10 mm groups. The BRAF^V600E mutation was associated with male sex (P = 0.028), large tumor size, extrathyroidal extension, central and lateral lymph node metastasis, and advanced tumor stage (P<0.0001).

Conclusion

The BRAF^V600E mutation was significantly associated with several poor clinicopathologic characteristics, but was not associated with sonographic features, regardless of tumor size. We recommend that patients with a thyroid nodule with any suspicious sonographic feature undergo preoperative BRAF^V600E testing for risk stratification and to guide the initial surgical approach in PTC.     

Regulating the response to targeted MEK inhibition in melanoma: Enhancing apoptosis in NRAS- and BRAF-mutant melanoma cells with Wnt/β-catenin activation

The limitations of revolutionary new mutation-specific inhibitors of BRAF^V600E include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAF^V600E by PLX4720 synergistically induces apoptosis across a spectrum of BRAF^V600E melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.     

Lkb1 Loss Promotes Tumor Progression of BRAFV600E-Induced Lung Adenomas

Aberrant activation of MAP kinase signaling pathway and loss of tumor suppressor LKB1 have been implicated in lung cancer development and progression. Although oncogenic KRAS mutations are frequent, BRAF mutations (BRAF^V600E) are found in 3% of human non-small cell lung cancers. Contrary to KRAS mutant tumors, BRAF^V600E-induced tumors are benign adenomas that fail to progess. Interestingly, loss of tumor supressor LKB1 coexists with KRAS oncogenic mutations and synergizes in tumor formation and progression, however, its cooperation with BRAF^V600E oncogene is unknown. Our results describe a lung cell population in neonates mice where expression of BRAF^V600E leads to lung adenoma development. Importantly, expression of BRAF^V600E concomitant with the loss of only a single-copy of Lkb1, overcomes senencence–like features of BRAF^V600E-mutant adenomas leading malignization to carcinomas. These results posit LKB1 haploinsufficiency as a risk factor for tumor progression of BRAF^V600E mutated lung adenomas in human cancer patients.     

The BRAFV600E causes widespread alterations in gene methylation in the genome of melanoma cells

Although BRAF^V600E is well known to play an important role in the tumorigenesis of melanoma, its molecular mechanism, particularly the epigenetic aspect, has been incompletely understood. Here, we investigated the role of BRAF^V600E signaling in altering gene methylation in the genome of melanoma cells using a methylated CpG island amplification/CpG island microarray system and searched for genes coupled to the BRAF^V600E signaling through methylation aberrations. The results indicated that a wide range of genes with broad functions were linked to BRAF^V600E signaling through their hyper- or hypomethylation. Expression of 59 genes hypermethylated upon BRAF knockdown was selectively tested and found to be largely correspondingly underexpressed, suggesting that these genes were naturally hypomethylated and overexpressed with BRAF^V600E in melanoma. This BRAF^V600E-promoted hypomethylation was confirmed on genes selectively examined in primary melanoma tumors. Some of these genes were functionally tested and demonstrated to play a role in melanoma cell proliferation and invasion. As a mechanism of aberrant gene methylation driven by BRAF^V600E, expression of the DNA methyltransferase 1 and histone methyltransferase EZH2 was profoundly affected by BRAF^V600E. We have thus uncovered a previously unrecognized prominent epigenetic mechanism in the tumorigenesis of melanoma driven by BRAF^V600E. Many of the functionally important genes controlled by the BRAF^V600E signaling through aberrant methylation may prove to be novel therapeutic targets for melanoma.Key words: BRAF mutation, DNA methylation, melanoma, MAP kinase pathway, gene hypomethylation, gene hypermethylation     

The Relationship of BrafV600E Mutation Status to FDG PET/CT Avidity in Thyroid Cancer: A Review and Meta-Analysis

Objective: Papillary thyroid cancer (PTC) harboring a BRAF^V600E gene mutation has been shown to exhibit aggressive tumor behavior and carries higher risks of recurrence and disease-specific death. In this systematic review and meta-analysis, we examined published evidence related to the accuracy of fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) in detection of residual disease in patients with BRAF^V600E mutated thyroid cancer.Methods: We extracted data from PUBMED/MEDLINE and EMBASE published between January 1995 and March 2017. We included studies that compared FDG PET standardized uptake values (SUVs) between BRAF^V600E-positive and BRAF^V600E-negative subjects, as well as those that evaluated the odds of having FDG avidity between BRAF^V600E-positive and -negative patients with thyroid cancer.Results: There were a total of 12 studies in the systematic review. Seven studies qualified for the analysis for calculating the pooled odds ratio (OR). The pooled cohort with binary data had 1,144 patients out of which 843 were BRAF^V600E positive and 301 were BRAF^V600E negative. Those with a BRAF^V600E mutation had a significantly greater likelihood of having FDG-avid lesions. The pooled OR was 2.12 (confidence interval &lsqb;CI] 1.53–3.00, P<.01). The pooled mean SUV (cohort of 315 patients) was significantly higher in BRAF^V600E-positive compared to BRAF^V600E negative patients, with a pooled mean difference of 5.1 (CI 4.3–5.8).Conclusion: Our meta-analysis shows that presence of BRAF^V600E mutation in PTC confers a higher likelihood of FDG PET avidity and is associated with higher SUV uptake values compared to BRAF^V600E-mutation negative status.Abbreviations: BRAF = B-Raf proto-oncogene, serine/threonine kinase; CI = confidence interval; CT = computed tomography; DTC = differentiated thyroid cancer; FDG = fluorodeoxyglucose; PET = positron emission tomography; PTC = papillary thyroid cancer; SUV = standardized uptake value     

The BRAFV600E causes widespread alterations in gene methylation in the genome of melanoma cells

Although BRAF^V600E is well known to play an important role in the tumorigenesis of melanoma, its molecular mechanism, particularly the epigenetic aspect, has been incompletely understood. Here, we investigated the role of BRAF^V600E signaling in altering gene methylation in the genome of melanoma cells using a methylated CpG island amplification/CpG island microarray system and searched for genes coupled to the BRAF^V600Esignaling through methylation aberrations. The results indicated that a wide range of genes with broad functions were linked to BRAF^V600E signaling through their hyper- or hypomethylation. Expression of 59 genes hypermethylated upon BRAF knockdown was selectively tested and found to be largely correspondingly underexpressed, suggesting that these genes were naturally hypomethylated, and overexpressed with BRAF^V600E in melanoma. This BRAF^V600E-promoted hypomethylation was confirmed on genes selectively examined in primary melanoma tumors. Some of these genes were functionally tested and demonstrated to play a role in melanoma cell proliferation and invasion. As a mechanism of aberrant gene methylation driven by BRAF^V600E, expression of the DNA methyltransferase 1 and histone methyltransferase EZH2 was profoundly affected by BRAF^V600E. We have thus uncovered a previously unrecognized prominent epigenetic mechanism in the tumorigenesis of melanoma driven by BRAF^V600E. Many of the functionally important genes controlled by the BRAF^V600E signaling through aberrant methylation may prove to be novel therapeutic targets for melanoma.     

The BRAF mutation confers sensitivity of thyroid cancer cells to the BRAF inhibitor PLX4032 (RG7204)

Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF^V600E, as a result of the BRAF^T1799A mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF^V600E-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF^T1799A mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF^T1799A mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC₅₀ values (0.115–1.156 μM) in BRAF^V600E mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC₅₀ values (56.674–1349.788 μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF^T1799A mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF^T1799A mutation-selective therapeutic agent for thyroid cancer.     

Tumorigenesis of Papillary Thyroid Cancer Is Not BRAF-Dependent in Patients with Acromegaly

Introduction

Several studies have reported a high frequency of papillary thyroid cancer (PTC) in patients with acromegaly. The aim of this study was to determine the prevalence and predictors of thyroid cancer in patients with acromegaly and to investigate the frequency of the BRAF ^V600E mutation in PTC patients with and without acromegaly.

Materials and Methods

We conducted a retrospective study of 60 patients with acromegaly. Thyroid ultrasonography (US) and US-guided fine needle aspiration were performed on nodules with sonographic features of malignancy. We selected 16 patients with non-acromegalic PTC as a control group. The BRAF ^V600E mutation was analyzed in paraffin-embedded surgical specimens of PTC by real-time polymerase chain reaction, and tumor specimens from patients with PTC were stained immunohistochemically with an antibody against insulin-like growth factor-1 receptor β (IGF-1Rβ).

Results

Thyroid cancer was found in 15 (25.0%) patients. No differences in age, sex, initial growth hormone (GH) and IGF-1 percentage of the upper limit of normal values or treatment modalities were observed between patients with and without PTC. Acromegaly was active in 12 of 15 patients at the time of PTC diagnosis; uncontrolled acromegaly had a significantly higher frequency in the PTC group (60%) than in the non-PTC group (28.9%) (p = 0.030). The BRAF ^V600E mutation was present in only 9.1% (1/11) of PTC patients with acromegaly, although 62.5% (10/16) of control patients with PTC had the mutation (p = 0.007). IGF-1Rβ immunostaining showed moderate-to-strong staining in all malignant PTC cells in patients with and without acromegaly. Significantly less staining for IGF-1Rβ was observed in normal adjacent thyroid tissues of PTC patients with acromegaly compared with those without (p = 0.014).

Conclusion

The prevalence of PTC in acromegalic patients was high (25%). An uncontrolled hyperactive GH-IGF-1 axis may play a dominant role in the development of PTC rather than the BRAF ^V600E mutation in patients with acromegaly.     

LncRNA HOTAIR regulates the expression of E-cadherin to affect nasopharyngeal carcinoma progression by recruiting histone methylase EZH2 to mediate H3K27 trimethylation

Background/AimThere has been increasing evidence for the function of long non-coding RNA (lncRNA) in nasopharyngeal carcinoma (NPC). We aim to delve into the position of lncRNA HOX antisense intergenic RNA (HOTAIR), together with enhancer of zeste homolog 2 (EZH2), E-cadherin and trimethylation of lysine 27 on histone H3 (H3K27me3) in NPC.MethodsHOTAIR, EZH2, and E-cadherin expression in NPC tissues and cells were tested. NPC cell biological functions were examined through gain-of and loss-of function assays. The mechanism of lncRNA HOTAIR/E-cadherin/EZH2/H3K27 axis in NPC was decoded.ResultsLncRNA HOTAIR and EZH2 were highly expressed in NPC, and E-cadherin was lowly expressed. Down-regulation of HOTAIR or EZH2 inhibited NPC cell progression and tumor growth. HOTAIR recruited histone methylase EZH2 to mediate trimethylation of H3K27 and regulated E-cadherin expression.ConclusionHOTAIR inhibits E-cadherin by stimulating the trimethylation of H3K27 to promote NPC cell progression through recruiting histone methylase EZH2.     

Identification of novel 3,5-diarylpyrazoline derivatives containing salicylamide moiety as potential anti-melanoma agents

There is an accumulating body of experimental evidences validating oncogenic BRAF^V600E as a therapeutic target and offering opportunities for anti-melanoma drug development. Encouraged by the positive results of pyrazole derivatives as BRAF^V600E inhibitors, we sought to design diverse novel potential BRAF^V600E inhibitors as antitumor agents based on pyrazole skeleton. In silico and in vitro screening of our designed pyrazole derivatives has identified Hit 1 as BRAF^V600E inhibitor. Based on its structure and through further structure modification, compound 25, which exhibited the most potent inhibitory activity with an IC₅₀ value of 0.16 μM for BRAF^V600E and GI₅₀ value of 0.24 μM for mutant BRAF-dependent melanoma cells, was obtained. The 3D-QSAR models and the molecular docking simulation were introduced to analyze the structure–activity relationship.     

Tumor Homogeneity between Primary and Metastatic Sites for BRAF Status in Metastatic Melanoma Determined by Immunohistochemical and Molecular Testing

BRAF inhibitors have demonstrated improvement of overall survival in patients with metastatic melanoma and BRAF^V600 mutations. In order to evaluate BRAF tumor heterogeneity between primary and metastatic site, we have evaluated the performance of immunohistochemistry (IHC) with an anti-BRAF^V600E antibody in both localization by comparison with high resolution melting analysis followed by Sanger sequencing in a parallel blinded study. A total of 230 samples distributed as primary melanoma (n = 88) and different types of metastatic samples (n = 142) were studied in 99 patients with advanced or metastatic melanoma (stage III or IV). The prevalence of each BRAF mutation was c.1799T>A, BRAF^V600E (45.2%), c.1799_1800TG>AA, BRAF^V600E2 (3.0%), c.1798_1799GT>AA, BRAF^V600K (3.0%), c.1801 A>G, BRAF^K601E (1.3%), c.1789_1790CT>TC, BRAF^L597S (0.4%), c.1780G>A, BRAF^D594N (0.9%) respectively. IHC was positive in 109/112 samples harboring BRAF^V600E/E2 mutations and negative in other cases. The cytoplasmic staining was either strongly positive in tumor cells of BRAF^V600E mutated cases. It appeared strong brown, different from the vesicular grey cytoplasmic pigmentation of melanophages. Concordance between the two techniques was 96.4%. Sensitivity of IHC for detecting the BRAF^V600E/E2 mutations was 97.3%, while specificity was 100%. Both our IHC and molecular study demonstrated homogeneity between primary and metastatic sites for BRAF status in melanoma. This study also provides evidence that IHC may be a cost-effective first-line method for BRAF^V600E detection. Thereafter, molecular techniques should be used in negative, ambiguous or non-contributive cases.     

Early differential responses elicited by BRAFV600E in adult mouse models

The BRAF gene is frequently mutated in cancer. The most common genetic mutation is a single nucleotide transition which gives rise to a constitutively active BRAF kinase (BRAF^V600E) which in turn sustains continuous cell proliferation. The study of BRAF^V600E murine models has been mainly focused on the role of BRAF^V600E in tumor development but little is known on the early molecular impact of BRAF^V600E expression in vivo. Here, we study the immediate effects of acute ubiquitous BRAF^V600E activation in vivo. We find that BRAF^V600E elicits a rapid DNA damage response in the liver, spleen, lungs but not in thyroids. This DNA damage response does not occur at telomeres and is accompanied by activation of the senescence marker p21^CIP1 only in lungs but not in liver or spleen. Moreover, in lungs, BRAF^V600E provokes an acute inflammatory state with a tissue-specific recruitment of neutrophils in the alveolar parenchyma and macrophages in bronchi/bronchioles, as well as bronchial/bronchiolar epithelium transdifferentiation and development of adenomas. Furthermore, whereas in non-tumor alveolar type II (ATIIs) pneumocytes, acute BRAF^V600E induction elicits rapid p53-independent p21^CIP1 activation, adenoma ATIIs express p53 without resulting in p21^CIP1 gene activation. Conversely, albeit in Club cells BRAF^V600E-mediated proliferative cue is more exacerbated compared to that occurring in ATIIs, such oncogenic stimulus culminates with p21^CIP1-mediated cell cycle arrest and apoptosis. Our findings indicate that acute BRAF^V600E expression drives an immediate induction of DNA damage response in vivo. More importantly, it also results in rapid differential responses of cell cycle and senescence-associated proteins in lung epithelia, thus revealing the early molecular changes emerging in BRAF^V600E-challenged cells during tumorigenesis in vivo.Subject terms: Lung cancer, Senescence     

Design,synthesis, and biological evaluation of pyrazole derivatives containing acetamide bond as potential BRAFV600E inhibitors

With the increasingly acquired resistance, relapse and side effects of known marketed BRAF^V600E inhibitors, it’s significant to design the more effective and novel drugs. In this study, a series of novel pyrazole derivatives containing acetamide bond had been designed and synthesized on the basis of analysis of the endogenous ligands extracted from the known B-Raf co-crystals in the PDB database. Then, the compounds were evaluated for biological activities as potential BRAF^V600E inhibitors. The bioassay results in vitro against three human tumor cell lines revealed that some of the compounds showed very impressed antiproliferative property. Among them, the compound 5r with IC₅₀ values of 0.10?±?0.01?μM against BRAF^V600E and 0.96?±?0.10?μM against A375 cell line, showed the most potent inhibitory effect, compared with the positive-controlled agents vemurafenib (IC₅₀?=?0.04?±?0.004?μM for BRAF^V600E, IC₅₀?=?1.05?±?0.10?μM against A375). Further investigation confirmed that the compound 5r could induce A375 cell apoptosis, induce A375 cell death through changing mitochondrial membrane potential, and result in A375 cell arrest at the G1 phase of the cell cycle. Docking simulations results indicated that the compound 5r could bind tightly at the BRAF^V600E active site. Meanwhile, 3D-QSAR model suggested that these compounds may be potential anticancer inhibitors. Overall, the article provided some new molecular scaffolds for the further BRAF^V600E inhibitors.