Characterization of conditionally targeted inhibitor of nuclear factor kappa B kinase subunit epsilon (IKBKE) in mutant mice

• Grant number: 21/09946-0
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Effective date (Start): February 01, 2022
• Effective date (End): January 31, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: José Cesar Rosa Neto
• Grantee: Loreana Sanches Silveira
• Supervisor: Jaswinder K. Sethi
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Research place: University Hospital Southampton NHS Foundation Trust, England
• Associated to the scholarship: 19/09679-2 - Characterization of PPARgamma knockout macrophages subpopulation in breast cancer model and the influence of moderate aerobic training, BP.PD

Abstract

Cancer is one of the main causes of death worldwide, and the leading type of cancer new cases in 2020 was breast cancer. Triple-negative breast cancer (TNBC), accounts for 10-15% of all breast cancers and is characterized by lack of estrogen, progesterone and human epidermal growth factor receptors expression. It is the most aggressive, and therefore therapies are more intricate. Immune cells, including monocytes are recruited into the tumour, differentiate and become tumour-associated macrophages (TAMs) which play an important role in tumour progression and metastasis. The peroxisome proliferator-activated receptor gamma (PPAR³) and the nuclear factor kappa B (NFºB) are two transcription factors that have opposite actions. PPAR³expression is associated with a pro tumorigenic environment. However, the molecular mechanisms behind the anti proliferative effects of PPAR³ in breast cancers are still poorly understood. NFºBactivity promotes cell cycle while PPAR³ inhibits cell proliferation. Importantly, NFºB is known to negatively regulate PPAR³ activation and several mechanisms have suggested how this is achieved at the molecular level. Activation of NFºB involves phosphorylation of inhibitor of ºB proteins byIºB kinases (IKK). Inhibitor of kappa B kinase epsilon (IKBKE) is an oncogene a berrantly increased in TNBC. However, the role of IKBKE in immune cell or TAM survival and/or metabolism is not known. Objective: The purpose of the present study is to validate and characterize theimmunometabolic profile and regulation of PPAR³ in a model lacking IKBKE in myeloid cells. Methods: Firstly, IKBKE ablation in myeloid cells - specifically splenocytes and bone marrowdifferentiated macrophages (BMDM) - will be confirmed by cell markers using FACs, protein(western blot) and/or gene (real time-PCR) analysis. Then, metabolic parameters and PPAR³expression will be analysed in conditional IKBKE knockout macrophages by Seahorse and proteinexpression (western blot). Whole tissue location (spleen, liver, adipose tissue, gut and skin) ofIKBKE and PPAR³ protein will be determined by immunohistochemistry. Co-culture of BMDMfrom IKBKE knockout macrophages using breast cancer cells conditioned media with PPAR³agonist and antagonist will be done to understand the complementary role of both IKBKE and PPAR³.Finally, IKBKE expression from paraffin-embedded TNBC samples from PPAR³ conditionalknockout macrophages will be verified by immunohistochemistry and 3D imaging. (AU)