Pharmacological levels of withaferin A (Withania somnifera) trigger clinically relevant anticancer effects specific to triple negative breast cancer cells

Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer

Cessação da coisa julgada nas relações tributárias de trato sucessivo: análise dos fundamentos determinantes do julgamento dos Temas 881 e 885 de repercussão geral pelo Supremo Tribunal Federal

Este trabalho tem como finalidade abordar, à luz dos princípios constitucionais e processuais que regem a matéria, os principais fundamentos que levaram ao julgamento dos Temas 881 (RE 949.297/CE) e 885 (RE 955.227/BA) de Repercussão Geral pelo Supremo Tribunal Federal, nos quais restou fixada a seguinte tese: “1. As decisões do STF em controle incidental de constitucionalidade, anteriores à instituição do regime de repercussão geral, não impactam automaticamente a coisa julgada que se tenha formado, mesmo nas relações jurídicas tributárias de trato sucessivo. 2. Já as decisões proferidas em ação direta ou em sede de repercussão geral interrompem automaticamente os efeitos temporais das decisões transitadas em julgado nas referidas relações, respeitadas a irretroatividade, a anterioridade anual e a noventena ou a anterioridade nonagesimal, conforme a natureza do tributo.” Diante disso, a primeira problemática a ser tratada refere-se a uma análise do instituto da coisa julgada e das suas implicações, vale dizer, da noção de segurança jurídica a ele atrelada e de sua indispensabilidade ao próprio Estado Democrático de Direito. Ainda, abordar-se-á sua aplicação a relações de trato sucessivo, e suas particularidades nesse contexto. Seguindo nessa linha, desenvolver-se-á considerações referentes ao controle de constitucionalidade e à repercussão geral no Direito Brasileiro, principalmente suas razões de ser e seus efeitos quando considerado o sistema de precedentes introduzido pelo Código de Processo Civil de 2015. Posteriormente, serão comentadas as circunstâncias do julgamento dos Temas 881 e 885/STF, quais sejam: origem da discussão, objeto do julgamento e fundamentos das decisões. Aqui, dedicar-se-á uma parcela da pesquisa a diferenciar a controvérsia envolvida nos dois Recursos Extraordinários supracitados daquela julgada no RE 730.462/SP (Tema 733/STF), que também configura precedente vinculante. Por fim, serão analisadas as implicações do julgamento dos Temas 881 e 885/STF, principalmente aquelas referentes às supostas desestabilização da coisa julgada e violação dos princípios constitucionais que regem o processo civil, bem como do cabimento da modulação dos efeitos das decisõesThe present work aims to address, based on the constitutional and procedural principles that govern the subject, the main arguments that were used on the judgement of Themes 881 (Extraordinary Appeal n. 949.297/CE) and 885 (Extraordinary Appeal n. 955.227/BA) of General Repercussion by the Supreme Federal Court, in which the following thesis was established: “1. The Supreme Federal Court’s decisions on incidental control of constitutionality, rendered before the institution of the general repercussion regimen, do not automatically impact the already formed ‘res judicata’, even on tax relations of successive tract. 2. On the other hand, decisions rendered on direct actions or on general repercussion automatically interrupt the temporal effects of final and unappealable decisions in those relations, respected the non-retroactivity, the annual anteriority and the 90-day holding period, according to the nature of the tax.” Therefore, the first problematic to be addressed refers to the institute of the res judicata and its implications, is to say, the notion of legal certainty that is attached to it and its indispensability to the Legal Democratic State. Also, its application to relations of successive tract, specifically on tax matters, will be approached, as well as its particularities in that specific context. Following that line, considerations will be developed regarding the constitutionality control and the general repercussion regimens, mainly concerning the reasons for their existence and their effects when taken into account the system of precedents introduced by the Civil Procedure Code of 2015. After that, the circumstances of the judgement of Themes 881 and 885 of General Repercussion will be addressed, them being: the origin of the discussions, the object of the judgement and the fundaments of the decisions. In this chapter, part of the research will be guided towards distinguishing the controversy involved in those judgements from the one involved in the judgement of Extraordinary Appeal 730.462/SP (Theme 733 of the Supreme Federal Court), which is also a binding precedent. Finally, the implications of the judgements will be examined, mainly those related to the (supposed) destabilization of the res judicata and violation of the constitutional principles that govern the civil procedure, as well as the need – or not – of an adjustment of the effects of the decision

CMS: A web-based system for visualization and analysis of genome-wide methylation data of human cancers

DNA methylation of promoter CpG islands is associated with gene suppression, and its unique genome-wide profiles have been linked to tumor progression. Coupled with high-throughput sequencing technologies, it can now efficiently determine genome-wide methylation profiles in cancer cells. Also, experimental and computational technologies make it possible to find the functional relationship between cancer-specific methylation patterns and their clinicopathological parameters.Cancer methylome system (CMS) is a web-based database application designed for the visualization, comparison and statistical analysis of human cancer-specific DNA methylation. Methylation intensities were obtained from MBDCap-sequencing, pre-processed and stored in the database. 191 patient samples (169 tumor and 22 normal specimen) and 41 breast cancer cell-lines are deposited in the database, comprising about 6.6 billion uniquely mapped sequence reads. This provides comprehensive and genome-wide epigenetic portraits of human breast cancer and endometrial cancer to date. Two views are proposed for users to better understand methylation structure at the genomic level or systemic methylation alteration at the gene level. In addition, a variety of annotation tracks are provided to cover genomic information. CMS includes important analytic functions for interpretation of methylation data, such as the detection of differentially methylated regions, statistical calculation of global methylation intensities, multiple gene sets of biologically significant categories, interactivity with UCSC via custom-track data. We also present examples of discoveries utilizing the framework.CMS provides visualization and analytic functions for cancer methylome datasets. A comprehensive collection of datasets, a variety of embedded analytic functions and extensive applications with biological and translational significance make this system powerful and unique in cancer methylation research. CMS is freely accessible at: http://cbbiweb.uthscsa.edu/KMethylomes/

Pubertal high fat diet: effects on mammary cancer development

INTRODUCTION: Epidemiological studies linking dietary fat intake and obesity to breast cancer risk have produced inconsistent results. This may be due to the difficulty of dissociating fat intake from obesity, and/or the lack of defined periods of exposure in these studies. The pubertal mammary gland is highly sensitive to cancer-causing agents. We assessed how high fat diet (HFD) affects inflammation, proliferative, and developmental events in the pubertal gland, since dysregulation of these can promote mammary tumorigenesis. To test the effect of HFD initiated during puberty on tumorigenesis, we utilized BALB/c mice, for which HFD neither induces obesity nor metabolic syndrome, allowing dissociation of HFD effects from other conditions associated with HFD. METHODS: Pubertal BALB/c mice were fed a low fat diet (12% kcal fat) or a HFD (60% kcal fat), and subjected to carcinogen 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumorigenesis. RESULTS: HFD elevated mammary gland expression of inflammatory and growth factor genes at 3 and 4 weeks of diet. Receptor activator of nuclear factor kappa-B ligand (RANKL), robustly induced at 4 weeks, has direct mitogenic activity in mammary epithelial cells and, as a potent inducer of NF-κB activity, may induce inflammatory genes. Three weeks of HFD induced a transient influx of eosinophils into the mammary gland, consistent with elevated inflammatory factors. At 10 weeks, prior to the appearance of palpable tumors, there were increased numbers of abnormal mammary epithelial lesions, enhanced cellular proliferation, increased growth factors, chemokines associated with immune-suppressive regulatory T cells, increased vascularization, and elevated M2 macrophages. HFD dramatically reduced tumor latency. Early developing tumors were more proliferative and were associated with increased levels of tumor-related growth factors, including increased plasma levels of HGF in tumor-bearing animals. Early HFD tumors also had increased vascularization, and more intra-tumor and stromal M2 macrophages. CONCLUSIONS: Taken together in this non-obesogenic context, HFD promotion of inflammatory processes, as well as local and systemically increased growth factor expression, are likely responsible for the enhanced tumorigenesis. It is noteworthy that although DMBA mutagenesis is virtually random in its targeting of genes in tumorigenesis, the short latency tumors arising in animals on HFD showed a unique gene expression profile, highlighting the potent overarching influence of HFD

Contribution of CXCL12 secretion to invasion of breast cancer cells

INTRODUCTION: Neu (HER2/ErbB2) is overexpressed in 25% to 30% of human breast cancer, correlating with a poor prognosis. Researchers in previous studies who used the mouse mammary tumor virus Neu-transgenic mouse model (MMTV-Neu) demonstrated that the Neu-YB line had increased production of CXCL12 and increased metastasis, whereas the Neu-YD line had decreased metastasis. In this study, we examined the role of increased production of CXCL12 in tumor cell invasion and malignancy. METHODS: We studied invasion in the tumor microenvironment using multiphoton intravital imaging, in vivo invasion and intravasation assays. CXCL12 signaling was altered by using the CXCR4 inhibitor AMD3100 or by increasing CXCL12 expression. The role of macrophage signaling in vivo was determined using a colony-stimulating factor 1 receptor (CSF-1R) blocking antibody. RESULTS: The Neu-YD strain was reduced in invasion, intravasation and metastasis compared to the Neu-YB and Neu deletion mutant (activated receptor) strains. Remarkably, in the Neu-YB strain, in vivo invasion to epidermal growth factor was dependent on both CXCL12-CXCR4 and CSF1-CSF-1R signaling. Neu-YB tumors had increased macrophage and microvessel density. Overexpression of CXCL12 in rat mammary adenocarcinoma cells increased in vivo invasion as well as microvessel and macrophage density. CONCLUSIONS: Expression of CXCL12 by tumor cells results in increased macrophage and microvessel density and in vivo invasiveness

Loss of amphiregulin reduces myoepithelial cell coverage of mammary ducts and alters breast tumor growth

Abstract Background Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth. Methods We crossed AREG-null (AREG−/−) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG−/− PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors. Results Intriguingly, PyMT-induced lesions progress more rapidly in AREG−/− mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG−/− mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG−/− PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas. Conclusions Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer

Tau filament self-assembly and structure: tau as a therapeutic target

Tau plays an important pathological role in a group of neurodegenerative diseases called tauopathies, including Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy and corticobasal degeneration. In each disease, tau self-assembles abnormally to form filaments that deposit in the brain. Tau is a natively unfolded protein that can adopt distinct structures in different pathological disorders. Cryo-electron microscopy has recently provided a series of structures for the core of the filaments purified from brain tissue from patients with different tauopathies and revealed that they share a common core region, while differing in their specific conformation. This structurally resolvable part of the core is contained within a proteolytically stable core region from the repeat domain initially isolated from AD tau filaments. Tau has recently become an important target for therapy. Recent work has suggested that the prevention of tau self-assembly may be effective in slowing the progression of Alzheimer's disease and other tauopathies. Here we review the work that explores the importance of tau filament structures and tau self-assembly mechanisms, as well as examining model systems that permit the exploration of the mode of action of potential inhibitors

Metastatic small-cell lung carcinoma to the thyroid gland: a case report

AbstractObjectMetastasis to the thyroid gland is a rare event.MethodsWe describe an unusual case of a 38-year-old woman with a history of small-cell lung cancer (SCLC), presenting with a new nodule in the thyroid gland, found to be metastatic SCLC, without evidence of widespread dissemination.ConclusionsA new thyroid nodule should be carefully evaluated for metastasis in a patient with a history of prior malignancy.</jats:sec