New developments on the pathogenesis of systemic lupus erythemstosus

Tese de doutoramento, Medicina (Pediatria), Universidade de Lisboa, Faculdade de Medicina, 2018Systemic Lupus Erythematosus (SLE) is a challenging autoimmune disease, with a complex etiopathogenesis and an unpredictable clinical course. In a large cohort of juvenile-onset SLE patients, we found that major infections were common, were associated with active disease and its treatment, and resulted in noteworthy morbidity. New biomarkers to guide the judicious use of immunosuppressive drugs and new treatment strategies with fewer side effects would, therefore, have an enormous impact in the management of these patients. In order to reach these goals we used the modern tools of molecular biology and focused on two of the most important complications of SLE: lupus nephritis and macrophage activation syndrome. Firstly, we identified the kidney lupus nephritis specific microRNA (miRNA) signature, which reflected mainly cell proliferation. MiRNAs are noncoding RNAs responsible for post-transcriptional gene silencing. These key regulatory molecules control the expression of multiple genes, so its dysregulation can contribute to sustained pathology. We showed that miR-26a and miR-30b were significantly decreased in the kidneys and urine of lupus nephritis patients. In vitro, the knockdown of miR-26a and miR-30b caused the proliferation of human mesangial cells and increased the expression of genes related to the cell cycle, including CCNE2, E2F8, MAD2L1, MYBL1 and POLQ. The Human Epidermal Growth Factor Receptor 2 (HER2) is a protein previously known to regulate miR-26a and miR-30b expression. Trastuzumab, a monoclonal antibody against HER2, used in breast cancer treatment, produces therapeutic actions precisely by up-regulating miR-26a and miR-30b. In human mesangial cells we also found that trastuzumab increases these two miRNAs. We hypothesized that HER2 also played a role in the pathogenesis of lupus nephritis and indeed we identified a dramatic overexpression of HER2 in the glomeruli and tubular compartments of the kidneys of lupus nephritis patients. The same pattern was not seen in the kidneys of healthy individuals or in other proliferative glomerulonephritides, including post-streptococcus glomerulonephritis, IgA nephropathy and granulomatosis with polyangiitis. Furthermore, in the lupus-prone NZM2410 mice we identified a highly increased expression of HER2, which correlated with disease activity. Finally, we showed that urinary HER2 was significantly increased in lupus nephritis and that its levels increased during flares, were higher in class III and class IV lupus nephritis and correlated with urinary proteincreatinine and monocyte chemoattractant protein 1 (MCP1) and vascular cell adhesion protein 1 (VCAM1) levels. We, therefore, established a strong rationale to use trastuzumab to block HER2 and decrease cell proliferation and damage in lupus nephritis. Regarding the macrophage activation syndrome, we were interested in the characterization of hemophagocytes. These are activated macrophages that have engulfed other hematopoietic cells. Traditionally they have been associated with the development of cytopenias in several life-threatening cytokine storm syndromes. New data have challenged this concept, since pancytopenia occurs in the absence of hemophagocytosis in mice and, in humans, over 40% of patients with macrophage activation syndrome do not have hemophagocytes in bone marrow aspirates. On the other hand, subclinical hemophagocytosis is detected in more than 50% of patients with systemic juvenile idiopathic arthritis, but only 10% develop a life-threatening macrophage activation syndrome. Thus the function and significance of hemophagocytes remained mysterious. Recent evidence demonstrated that environmental factors, particularly the cytokine milieu, determine the macrophage activation status in a continuum ranging from M1 to M2. M1 macrophages, driven by interferon γ, typically acquire proinflammatory properties and are associated with tissue damage, whereas M2 macrophages have more heterogeneous stimuli and functions, being associated with immunoregulation, tissue remodeling and fibrosis. Since interferon γ is the hallmark cytokine of hemophagocytic lymphohistiocytosis, one would expect to find that hemophagocytes express M1 surface receptors. We characterized the transcriptional phenotype of mouse Toll-like receptor 9 (TLR9) – induced hemophagocytes and described the surface phenotype of human bone marrow hemophagocytes. Interestingly, murine hemophagocytes had up-regulation of genes associated with the M2 and not the M1 phenotype. Immunohistochemical analyses in bone marrow samples from a uniquely diverse cohort of patients with hemophagocytic syndromes showed universal staining of hemophagocytes for the M2 marker CD163, but rarely for CD206 or CD64. Collectively, these data support the hypothesis that hemophagocytes have immunoregulatory functions and open new doors for the study of the pathogenesis of this syndrome. Finally, we were interested in characterizing the transcriptome of SLE, by RNAsequencing, in order to better understand the mechanisms that are responsible for chronicity. Monocytes from SLE patients exhibited a globally dysregulated gene expression. The transcriptome was not simply altered by the activation of a set of genes, but was also qualitatively different. Splicing patterns and polyadenylation were significantly altered and SLE monocytes expressed novel transcripts, an effect that was replicated by exposing control monocytes to lipopolysaccharide (LPS). We further identified increased circulating endotoxin in SLE patients, suggesting that chronic microbial translocation could contribute to the immunologic dysregulation in SLE, a new potential disease mechanism. In conclusion, with these different projects, which globally focused on the transcriptome and epigenome of SLE, we identified novel pathways and challenged the current paradigms. We described new mechanisms of disease, including the role of LPS in transforming qualitatively the SLE transcriptome and a putative immunoregulatory function for hemophagocytes. We demonstrated that miR-26a, miR-30b and HER2 control cell proliferation in lupus nephritis and that are promising biomarkers. Most importantly, our work rose the possibility of using anti-HER2 drugs for lupus nephritis management, opening the door to a new treatment strategy in this disease.Fundação Calouste Gulbenkian, Fundação Champalimau

Genetic and molecular mechanisms of sarcomas

Sarcomas are heterogeneous malignant mesenchymal tumors with diverse biological features and unique clinical characteristics, the genetic alterations of sarcomas are highly variable. With the development of sequencing technologies, efficient and practical approaches to detect gene expressions and gene variants contribute to the prediction of patient prognosis and the choice of treatment modalities. Given the rarity of sarcomas, the comprehensive transcriptomic or genomic profiles are still lacking for many subtypes. In the present thesis, by applying sequencing technology in sarcoma cohorts, combined with bioinformatics data analysis and molecular biology experiments, we have revealed new biological mechanisms dictating sarcoma behavior and provided insights for clinical applications. In Paper I, we characterized the gene signatures related to poor prognosis, first-line treatment failure, and chemotherapy resistance in Ewing sarcoma (ES). High expression of IGF2 was associated with shorter overall survival in ES patients and promoted cell proliferation, radiation resistance, and apoptosis inhibition in vitro. The transcriptome analysis of clinical samples and cell lines uncovered an IGF-dependent signature and potentially related to stem cell-like signatures in ES. Paper II continued to highlight the transcriptome signatures in ES. Here, we identified prognosis-related RNA-binding proteins (RBPs) and constructed an RBP-based prognostic risk model that showed stable predictive power for evaluating overall survival in clinical samples. Within the model, NSUN7 is considered an independent prognostic favorable prognostic marker, which was also validated by immunohistochemistry. In Paper III, we discovered that TERT promoter mutations were present in 45% of patients in a cohort of 190 patients with conventional chondrosarcoma (CHS). The mutation was significantly associated with recurrence, distant metastasis, and high tumor grade. The heterogeneity of primary tumors and the altered mutational status between asynchronous metastatic lesions revealed that CHS is a multiclonal disease that progresses through branching evolution. In Paper IV, we identified three clusters with distinct transcriptomic and genomic patterns in synovial sarcoma (SS), of which SS cluster I (SSC-I) was characterized by hyperproliferation, immune cell silencing, and poor prognosis; SSC-II was characterized by high vascularity and stromal component with the better clinical outcome; SSC-III was characterized by epithelial components with genomic complexity and checkpoint-mediated immune suppression. Collectively, the present thesis illustrated the pathogenic mechanisms of ES, CHS, and SS through the analysis of transcriptomic and genomic data, identified prognostic biomarkers, and at the clinical application-level provided strong evidence for patient stratification, risk prediction, and personalized treatment assessment

Genomic instability in human cancer: molecular insights and opportunities for therapeutic attack and prevention through diet and nutrition

Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology

Translational Research in Cancer

Translational research in oncology benefits from an abundance of knowledge resulting from genome-scale studies concerning the molecular pathways involved in tumorigenesis. Translational oncology represents a bridge between basic research and clinical practice in cancer medicine. The vast majority of cancer cases are due to environmental risk factors. Many of these environmental factors are controllable lifestyle choices. Experimental cancer treatments are studied in clinical trials to compare the proposed treatment to the best existing treatment through translational research. The key features of the book include: 1) New screening for the development of radioprotectors: radioprotection and anti-cancer effect of β-Glucan (Enterococcus faecalis) 2) Translational perspective on hepatocellular carcinoma 3) Brachytherapy for endometrial cancer 4) Discovery of small molecule inhibitors for histone methyltransferases in cance

The Role of Glucocorticoid Signaling in Prostate Cancer Health Disparities

African-American men are more likely to develop aggressive prostate cancer (PCa) and die from the disease than other ethnic groups. Glucocorticoid signaling is a contributing biological factor to worse PCa prognosis, and is emerging as a key driver of PCa progression in the absence of androgens. The mechanism involves glucocorticoids binding to glucocorticoid receptor (GR) and bypassing the androgen receptor (AR) signaling pathway to activate AR-target genes that promote tumor aggressiveness and therapy-resistance. This is problematic as African-American men have hypersensitive GR signaling and chronically-elevated levels of glucocorticoids linked to cumulative stressful life events. To explore the role of glucocorticoid signaling in PCa health disparities, this dissertation used a racially diverse pre-clinical model to examine the effects of GR activation on the expression of stress oncoproteins linked to tumor aggressiveness and therapy-resistance, specifically Lens Epithelium-Derived Growth Factor p75 (LEDGF/p75) and Clusterin (CLU). Results revealed a robust pattern of GR-induced upregulation of LEDGF/p75 and CLU in African-American (AA) PCa cells compared to European-American (EA) PCa cells. We also detected increased GR transcript expression in AA PCa tissues, compared to EA tissues, using Oncomine microarray datasets. In addition, a trend towards elevated circulating LEDGF/p75 and CLU was observed in sera of AA patient samples. Taken together, these findings provide an initial framework for understanding the contribution of GR signaling to PCa health disparities

Classification of Lymphomas and Hematological Neoplasia in the Era of Genomic Research

This Special Issue contains review articles focusing on several lymphoma entities included in the current WHO classification. The aim of the book is to guide the readers in understanding the evolution of lymphoma classification. The clinicopathological entities described in this issue have been analyzed through the molecular mechanisms involved in their pathogenesis

Molecular Biology of Selenium in Health and Disease

The trace mineral selenium is still regarded as one of the most interesting and health-beneficial elements. In addition to the Editorial containing a dedication to Dr. Leopold Flohé, this Special Issue contains 13 research articles and 8 reviews, with over 120 different contributors covering many of the most important subjects concerning the study of selenium. The articles address both selenium as well as selenoproteins and their molecular roles, providing important considerations regarding this trace element’s impact on human and animal health and disease

Glioma

The tittle 'Glioma - Exploring Its Biology and Practical Relevance' is indicative of its content. This volume contains 21 chapters basically intended to explore glioma biology and discussing the experimental model systems for the purpose. It is hoped that the present volume will provide supportive and relevant awareness and understanding on the fundamental advances of the subject to the professionals from any sphere interested about glioma