O papel da PTX3 na modulação do perfil de expressão de miRNAs e na liberação de vesículas extracelulares em modelo de câncer de mama triplo negativo

Abstract

According to estimates from the World Health Organization (WHO), the number of new cases of cancer worldwide is increasing. Breast cancer is the most common tumor among women, with the triple-negative subtype (TNBC) being the most aggressive and less responsive to treatment, partly due to the absence of receptors for estrogen, progesterone and HER2 (human epidermal growth factor), which are targets of chemotherapy. Therefore, it becomes important to investigate new therapeutic targets for TNBC. Pentraxin 3 (PTX3) is an acute phase protein whose expression mainly occurs in response to inflammatory stimuli, such as TNFA and IL1B cytokines. Changes in PTX3 levels are associated with the severity of various diseases. In breast cancer, PTX3 plays a dual role, acting in some cases as a tumor suppressor and in others as an oncogenic agent. The hypothesis of this work is that PTX3expression of is related to the deregulation of miRNAs and extracellular vesicles (EVs) in TNBC. In this sense, the present study aims to investigate the role of human recombinant PTX3 protein (rhPTX3) in modulating miRNAs expression and EVs release in an in vitro model of TNBC. Basal gene expression of PTX3 in the MDA-MB-231 cell line was evaluated by RT-qPCR as well as the kinetics of PTX3 and TNFA gene expression after treatment with the rhPTX3 for 3, 6, 12 and 24 hours. Next Generation Sequencing (NGS) of small RNAs from rhPTX3- treated or untreated MDA- MB-231 for 3 hours was performed to assess miRNAs modulation by rhPTX3 and the interaction networks of differentially expressed miRNAs (DEMs). EVs release by MDA-MB- 231 cells treated for 24 hours with rhPTX3 was also analyzed through nanoparticle tracking, flow cytometry and protein quantification. The results showed that PTX3 is constitutively expressed in the MDA-MB-231 breast adenocarcinoma cell line and that treatment with rhPTX3 leads to a significant increase in its expression, with peaking at 6 hours in the MDA- MB-231 cell line. . The results also showed that TNFA is not constitutively expressed in MDA- MB-231 cells nor in response to rhPTX3 treatment. PTX3 altered the gene expression profile of 142 miRNAs, with 112 being positively modulated and 30 negatively modulated. In silico analysis identified that 12,894 targets for DEMs, and 29 canonical pathways were enriched. In this initial phase, the study focused on 3 of these pathways: ERK/MAPK signaling, regulation of breast cancer by Stathmin1, and the tumor microenvironment pathway. Inhibition of molecules related to cancer inflammation (IL6 and IL4) molecules regulating chemotaxis (CXCL8, CXCR4 and CXCL12); activation of pro-apoptotic molecule (BAD), inhibition of anti-apoptotic molecule (BCL2), and inhibition of molecules related to cell adhesion (ICAM1, CD44 and CRK) were identified in these pathways. Biofunction analyzes in IPA software of DEMs identified 5 activities: cell signaling and cell-to-cell interaction, cell movement, cell survival, tissue injuries and organ abnormalities, and cell morphology. Finally, analyzes of EVs release showed that although there was an increase in EV release in the rhPTX3 treated group, there was a reduction in of CD44+ EVs release. The initial findings of this work showed that PTX3 modulates miRNAs expression profile and increases EV release, decreasing the CD44+ EV population, thus acting as a tumor suppressor protein in the TNBC lineage, thus acting as a tumor suppressor protein in the CMTN lineage. Giving the limited knowledge of TNBC molecular pathways, our results present PTX3 and its subsequent activities as potential therapeutic targets.