(T1330-10-55) TP53 Codon 72 Polymorphism Modulates Macrophage Polarization through Altered PI3K/Akt Signaling Pathways

Purpose: The purpose of this study is to determine the role of the most common p53 single nucleotide polymorphism (SNP) at codon 72, which encodes proline (P72) or arginine (R72), in the regulation of macrophage polarization.

Methods: Bone marrow-derived macrophages (BMDMs) from human p53 knock-in (Hupki) mice, in which exons 4-9 of the endogenous mouse p53 allele were replaced with the homologous human p53 gene sequence, carrying P72 and R72 variant were treated with lipopolysaccharide (LPS) to activate macrophages and induce proinflammatory M1 phenotype. Signaling pathways involved in macrophage activation were analyzed by Real-Time RT-PCR, Western blotting, and immunofluorescence. A highly selective Akt inhibitor MK-2206 (Selleckchem), at a concentration of 0.1 uM, was used 60 min. prior to LPS to determine the effect of Akt blockade in R72 macrophage polarization and signaling. Volumes of tumors generated by subcutaneous injections of tumor cells (TC1), mixtures of tumor cells and LPS-stimulated P72 macrophages (TC1+P72^LPS), or a mixture of tumor cells and LPS-stimulated R72 macrophages (TC1+R72^LPS) were measured every four days, with tumor volumes calculated as length× Width× Width/2.

Results: LPS stimulation of BMDMs led to a greater upregulation of genes traditionally linked to M1 phenotype (Socs1 and Nos2) in P72 compared to R72 macrophages (Fig 1a). Further, we examined the activation of PI3K/Akt as this pathway is essential for restricting proinflammatory (M1) and promoting anti-inflammatory (M2) responses in toll-like receptor (TLR4)-stimulated macrophages [1]. The phosphorylation of Ser 473 residue of Akt was increased to a greater extent in R72 vs. P72 macrophages upon LPS treatment (Fig 2a). FOXO3a is a tumor suppressor and longevity factor (2) and downstream target of Akt. Consistent with increased Akt activity in R72 macrophages, the phosphorylation of FOXO3a at Ser 253 residue was greater in these cells than P72 macrophages upon LPS stimulation. This phosphorylation inhibits FOXO3a transcriptional activity via a nucleus to cytoplasm shuttle (Fig 2a). NF-kB activation, nuclear translocation, and subsequent transcriptional regulation is a key to the induction of several pro-inflammatory genes in macrophages [3]. Cytoplasmic FOXO3a was demonstrated to inhibit NF-kB via direct binding to NF-kB in the cytoplasm and prevent its nuclear translocation [4]. Through confocal microscopy and immunofluorescence, we demonstrate increased cytoplasmic colocalization of FOXO3a and NF-kB upon LPS stimulation (Fig 2b). Western blotting further corroborated these data showing impairment of NF-kB nuclear translocation (Fig 3a). Following treatment with MK2206, we observed reduced FOXO3a Ser 253 phosphorylation and enhanced NF-kB nuclear translocation (Fig 3b). Similarly, we observed increased expression of M1 genes (Socs1 & Nos2) in R72 cells treated with MK2206, to the level observed in P72 macrophage (Fig 1b). The addition of LPS-stimulated P72 macrophages (TC1+P72^LPS) to tumor cells reduced tumor growth, in contrast to R72 macrophages (TC1+R72^LPS) in a syngeneic model of HPV-induced cancer (Fig. 4a and b).

Conclusion: The role of the most common p53 SNP at codon 72, in the regulation of the immune system, has not yet been thoroughly explored. Here, we report that macrophages carrying R72 variant are biased toward M2 phenotype through a mechanism that involved the altered NF-kB nuclear translocation. In R72-LPS stimulated macrophages NF-kB is partially restrained in the cytoplasm by phosphorylated FOXO3a (S253). FOXO3a phosphorylation is enhanced by p-AKT (S473). Studies in a mouse model of cancer confirm the impact of this polymorphism on macrophage function in vivo by demonstrating that R72-LPS stimulated macrophages lose the ability to reduce tumor growth.

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