Modulation of NF-kB signaling pathway and repercussions on epigenetic marks in adipose cells: role of Oncotherad®

Abstract

White adipose tissue (WAT) has a high plasticity, responding to different metabolic demands, diets and drugs. The main cell responsible for these responses is the mature adipocyte, but mesenchymal stem cells contained in the vascular stromal fraction of WAT (AdSCs) allow its expansion through differentiation into new adipocytes. The commitment of AdSCs to the adipogenesis process depends on epigenetic alterations that lead to the activation and silencing of specific genes. Histone 3 lysine 27 (H3K27) acts as a switch in gene expression, as its acetylation leads to gene transcription, while its trimethylation leads to gene silencing. Ezh2 is responsible for trimethylation (H3K27me3), while Crebbp and Ep300 (Cbp/p300) are responsible for acetylation (H3K27ac), under the action of H3K27 demethylases. It is known that in cancer the expression of Ezh2 is regulated by the NF-kB family of transcription factors, but little is known about the regulation of Ezh2 transcription in TAB. Binding sites for NF-kB are also present in the promoter region of Crebbp, Ep300 and demethylases genes. In obese individuals, there is a significant increase in TNF-a and circulating endotoxins, which leads to the activation of NF-kB in WAT, which is associated with low-grade chronic inflammation that curses with obesity. This inflammation is secondary to the disproportionate increase in WAT, macrophage infiltration, increase in Toll-like receptors (TLR), such as TLR4, plasma insulin and WAT -secreted pro-inflammatory cytokines (such as leptin and IL-6) elevation simultaneously with the decrease of adiponectin, building a favorable scenario for different types of cancer. It is important to emphasize that depending on the context, TLR4 activation can induce an inflammatory process and proliferation (canonical pathway) or anti-inflammatory response and cell death (noncanonical pathway). The synthetic nanoparticle recently developed by brazilian researchers registered as OncoTherad®, activates TLR4 receptors and the non-canonical pathway that increases signaling for interferon, presenting immunomodulatory and antitumor properties. The action of OncoTherad® in WAT is not known, nor in obese individuals, who present exacerbation of pro-inflammatory cytokines regulated by the TLR4 and NF-ºB pathway, infiltration of activated macrophages and remodeling of WAT. Elucidating its effects on WAT, the largest endocrine organ and regulator of systemic energy homeostasis, is essential for its use. Thus, this work aims to analyze whether NF-kB transcription factors alter the expression of H3K27 modifiers in human preadipocyte strains and in visceral WAT samples from obese patients, the impact of this change in the expression of the transcription factor Klf15, as well as the modulation of this pathway by oncotherad. Experimental strategies involve 1) human preadipocyte model (hWA A41) and stimulation with LPS and macrophage conditioned medium, chromatin immunoprecipitation (ChIP), enzyme inhibitors and inhibition by interfering RNA (siRNA), and changes in RNA expression transcription factors potentially regulated by H3K27me3 in preadipocytes, such as Klf15; 2) investigate whether the findings in cell lines can be replicated in adipose tissue samples from obese patients; 3) collect WAT from patients treated or not with OncoTherad® and analyze whether the modulation of the NF-kB pathway by the nanodrug alters the expression of histone modifiers. This work will bring an important contribution to the understanding of the effect of inflammation on the expression of histone modifiers and on the epigenetic signature of transcription factors that act on adipogenesis and adipocyte metabolism, as well as elucidating the effects of OncoTherad® on WAT, being relevant for choice (or not) of its use in obese cancer patients. (AU)