The role of HMGA1 protein in gastroenteropancreatic neuroendocrine tumors

Neuroendocrine tumors (NETs) are neoplasms derived from neuroendocrine cells. One of their main features is to often remain asymptomatic and clinically undetectable. High Mobility Group A (HMGA) proteins belong to a family of non-histone chromatinic proteins able to modulate gene expression through the interaction with DNA and transcription factors. They are overexpressed in most of the human malignancies, playing a critical role in carcinogenesis. However, their expression levels and their role in neuroendocrine carcinogenesis has not been exhaustively evaluated until now. Therefore, in this study, we have addressed the validity of using the expression of HMGA1 as a diagnostic marker and have investigated its role in NET carcinogenesis. The expression of HMGA1 has been evaluated by qRT-PCR and immunohistochemistry, using NET tissue microarrays, in a cohort of gastroenteropancreatic (GEP)-NET samples. The expression levels of HMGA1 have been then correlated with the main clinical features of NET samples. Finally, the contribution of HMGA1 overexpression to NET development has been addressed as far as the modulation of proliferation and migration abilities of NET cells is concerned. Here, we report that HMGA1 is overexpressed in GEP-NET samples, at both mRNA and protein levels, and that the silencing of HMGA1 protein expression interferes with the ability of NET cells to proliferate and migrate through the downregulation of Cyclin E, Cyclin B1 and EZH2. These results propose the HMGA proteins as new diagnostic and prognostic markers

Epi-Regulation of Cell Death in Cancer

How do organisms regulate the correct balance between the production of “new” cells and the elimination of the “old” ones, remains an important biology issue under investigation. Cell(s) death represents a fundamental process involved in organism development and cell homeostasis, whose alteration is considered one hallmark of cancer and lead to drug resistance and consequently treatment failure. The recent re-classification of cell death has identified new molecular programs in which several proteins have a pivotal role. Several studies have highlighted a direct link between epigenetic modifications and cell death mechanisms. Different epi-modifications have been described, capable of regulating diverse key players implicated in cell death, leading to uncontrolled proliferation of cancer cells. Scientific efforts are focused on the understanding the epigenetic regulation of cell death mechanisms by developing tools and/or new epi-molecules able to overcome cell death resistance. The development of new epi-molecular tools can overcome cell death deregulation thus potentially improving the sensitivity to the anti-tumor therapies. This chapter focuses on the main epigenetic deregulations in cell death mechanisms in cancer

Enigmatic role of RIPK1 in leukemia

Introduction Cell survival, inflammation and death are essential physiological events for maintaining cellular homeostasis and for preventing disease, especially cancer. These processes may be regulated by receptor-interacting protein kinase 1 (RIPK1), whose activity depends on post-translational modifications [1]. While several advances in describing the molecular mechanisms involving RIPK1 are evident, the complex crosstalk in the regulation of its different functions in cancer makes it difficult to determine the precise events [1]. Although the activity of RIPK1 in a wide range of diseases and in tumorigenesis has been demonstrated, its role in leukemia is still highly debated and therefore unclear [2]. The heterogeneity of RIPK1 expression in leukemia patients and its enigmatic functions suggest to rapidly define the molecular mechanisms in hematologic malignancies [3]. Materials and methods To study the degree of RIPK1 expression in several human leukemia cell lines we performed Western blot and qRT-PCR analyses. To gain further mechanistic insights, RIPK1 expression was evaluated in nutrient deficiency (% serum in medium). The proteasome inhibitor MG132 was used to define the molecular mechanism. More sensitive detection methods (Immunoprecipitation and biosensors) have been used to assess the relative amount of RIPK1. Results RIPK1 protein in different leukemia cell models shows variable expression levels independent of its constant mRNA levels. Notably, despite the slight increase in PI positivity or alteration in cell cycle phases, U937 cells displayed divergent expression of RIPK1 protein after cell dilution and stress conditions. Indeed, nutrient deficiency downregulates RIPK1 which is restored after MG132 treatment. Furthermore, the more sensitive detection methods confirmed a reduced concentration of the target protein. Conclusions Our experiments confirm the variability of RIPK1 protein expression in leukemia and underline the hypothesis of a stress sensor whose mechanism depends on proteasome activity. Thus, these preliminary experiments are ideal for increasing our understanding of the transcriptomic and proteomic events underlying important biological processes that are not yet well characterized in leukemia

Re-Punching Tissue Microarrays Is Possible: Why Can This Be Useful and How to Do It

Tissue microarray (TMA) methodology allows the concomitant analysis of hundreds of tissue specimens arrayed in the same manner on a recipient block. Subsequently, all samples can be processed under identical conditions, such as antigen retrieval procedure, reagent concentrations, incubation times with antibodies/probes, and escaping the inter-assays variability. Therefore, the use of TMA has revolutionized histopathology translational research projects and has become a tool very often used for putative biomarker investigations. TMAs are particularly relevant for large scale analysis of a defined disease entity. In the course of these exploratory studies, rare subpopulations can be discovered or identified. This can refer to subsets of patients with more particular phenotypic or genotypic disease with low incidence or to patients receiving a particular treatment. Such rare cohorts should be collected for more specific investigations at a later time, when, possibly, more samples of a rare identity will be available as well as more knowledge derived from concomitant, e.g., genetic, investigations will have been acquired. In this article we analyze for the first time the limits and opportunities to construct new TMA blocks using tissues from older available arrays and supplementary donor blocks. In summary, we describe the reasons and technical details for the construction of rare disease entities arrays

HAT1: Landscape of Biological Function and Role in Cancer

Histone modifications, as key chromatin regulators, play a pivotal role in the pathogenesis of several diseases, such as cancer. Acetylation, and more specifically lysine acetylation, is a reversible epigenetic process with a fundamental role in cell life, able to target histone and non-histone proteins. This epigenetic modification regulates transcriptional processes and protein activity, stability, and localization. Several studies highlight a specific role for HAT1 in regulating molecular pathways, which are altered in several pathologies, among which is cancer. HAT1 is the first histone acetyltransferase discovered; however, to date, its biological characterization is still unclear. In this review, we summarize and update the current knowledge about the biological function of this acetyltransferase, highlighting recent advances of HAT1 in the pathogenesis of cancer

NETosis in pathologies: a preliminary study for NETs detection in vitro

Background: Neutrophils are the major participants in NETosis, a novel kind of regulated cell death (RCD) that has recently emerged. As a result, neutrophils not only serve as the initial line of defense for the host, but they also help to mediate the new RCD by releasing neutrophil extracellular traps (NETs).1 NETosis is characterized by sequence of events: intracellular membranes disintegrate and proteases from granules enter the nucleus, followed by hypercitrullination of histones, chromatin decondensation and extrusion of nuclear material from the cell. Then, NETs decorated by decondensed chromatin, modified histones and granular enzymes are released from cells. 2 Physiologically, NETs entrap bacteria and provide a natural defence against inflammation but an exacerbated release of NETs markers can exert pro-thrombotic and pro-inflammatory effects and are resulted implicated in many diseases such as hyperglycaemia, diabetes and its complications. 3 Aim: The project has the purpose to to study in depth NETosis pathway and its implications in pathogenesis. Specifically, will be characterize the epi-modulation of NETs formation in samples derived from cancer and diabetic patients. Materials and methods: differentiation of HL60 FOR 5 days with Dimethyl sulfoxide or All-trans retinoic acid. May Grunwald Giemsa staining. Immunofluorescence staining Anti-MPO and H3cit. Flow based assay to detect NETs. ROS assay. Result: several methods have been developed o investigate NETosis. NETs formation has been identified after epi drugs induction. The methods we are using to detect NETs and to evaluate NETosis markers are efficient to study NETosis in blood samples from patients. Conclusions: NETosis is a recent discovered RCD that resulted de-regulated in many pathologies. The characterization of NETosis mechanism and complete understanding of NETosis role in pathogenesis could provide new prognostic markers and novels therapeutic targets

Differential cell cycle and proliferation marker expression in ductal pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia (PanIN)

Pancreatic cancer is an aggressive tumour following a multistep progression model through precursors called pancreatic intraepithelial neoplasia (PanIN). Identification of reliable prognostic markers would help in improving survival. The aim of this study was to investigate the role as well as the prognostic significance of different cell cycle and proliferation markers, namely p21, p27, p53 and Ki-67, in pancreatic carcinogenesis