Non-small-cell lung carcinoma (NSCLC) is the major type of lung cancer, which is among the leading causes of cancer-related deaths worldwide. LIMD1 was previously identified as a tumor suppressor in lung cancer, but their detailed interaction in this setting remains unclear. In this study, we have carried out multiple genome-wide bioinformatic analyses for a comprehensive understanding of LIMD1 in NSCLC, using various online algorithm platforms that have been built for mega databases derived from both clinical and cell line samples. Our results indicate that LIMD1 expression level is significantly downregulated at both mRNA and protein levels in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), with a considerable contribution from its promoter methylation rather than its gene mutations. The Limd1 gene undergoes mutation only at a low rate in NSCLC (0.712%). We have further identified LIMD1-associated molecular signatures in NSCLC, including its natural antisense long non-coding RNA LIMD1-AS1 and a pool of membrane trafficking regulators. We have also identified a subgroup of tumor-infiltrating lymphocytes, especially neutrophils, whose tumor infiltration levels significantly correlate with LIMD1 level in both LUAD and LUSC. However, a significant correlation of LIMD1 with a subset of immune regulatory molecules, such as IL6R and TAP1, was only found in LUAD. Regarding the clinical outcomes, LIMD1 expression level only significantly correlates with the survival of LUAD (p<0.01) but not with that of LUSC (p>0.1) patients. These findings indicate that LIMD1 plays a survival role in LUAD patients at least by acting as an immune regulatory protein. To further understand the mechanisms underlying the tumor-suppressing function of LIMD1 in NSCLC, we show that LIMD1 downregulation remarkably correlates with the deregulation of multiple pathways that play decisive roles in the oncogenesis of NSCLC, especially those mediated by EGFR, KRAS, PIK3CA, Keap1, and p63, in both LUAD and LUSC, and those mediated by p53 and CDKN2A only in LUAD. This study has disclosed that LIMD1 can serve as a survival prognostic marker for LUAD patients and provides mechanistic insights into the interaction of LIMD1 with NSCLC, which provide valuable information for clinical applications.

Oxidation of polyunsaturated fatty acids contributes to different aspects of the inflammatory response due to the variety of products generated. Specifically, the oxidation of DHA produces the end-product, carboxyethylpyrrole (CEP), which forms a covalent adduct with proteins via an ϵ-amino group of lysines. Previously, we found that CEP formation is dramatically increased in inflamed tissue and CEP-modified albumin and fibrinogen became ligands for αDβ2 (CD11d/CD18) and αMβ2 (CD11b/CD18) integrins. In this study, we evaluated the effect of extracellular matrix (ECM) modification with CEP on the adhesive properties of M1-polarized macrophages, particularly during chronic inflammation. Using digested atherosclerotic lesions and in vitro oxidation assays, we demonstrated the ability of ECM proteins to form adducts with CEP, particularly, DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin, but not with collagen I. Using integrin αDβ2-transfected HEK293 cells, WT and αD−/− mouse M1-polarized macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading when compared with natural ECM ligands such as collagen IV, laminin, and fibrinogen. Integrin αDβ2 is critical for M1 macrophage adhesion to CEP. Based on biolayer interferometry results, the isolated αD I-domain demonstrates markedly higher binding affinity to CEP compared to the “natural” αDβ2 ligand fibrinogen. Finally, the presence of CEP-modified proteins in a 3D fibrin matrix significantly increased M1 macrophage retention. Therefore, CEP modification converts ECM proteins to αDβ2-recognition ligands by changing a positively charged lysine to negatively charged CEP, which increases M1 macrophage adhesion to ECM and promotes macrophage retention during detrimental inflammation, autoimmunity, and chronic inflammation.

The accumulation of pro-inflammatory macrophages in the inflamed vascular wall is a critical step in atherogenesis. The mechanism of macrophage retention within the site of inflammation is not understood yet. High adhesion that prevents macrophage migration is one of the potential mechanisms. We previously showed that integrin α D β 2 is upregulated on pro-inflammatory macrophages, promotes macrophage retention, and contributes to atherogenesis. However, we have not identified a key ligand for α D β 2 within the tissue, since α D β 2 does not interact with major ECM proteins, collagens, and laminins. We recently found that during acute inflammation, the oxidation of docosahexaenoic acid (DHA) leads to the generation of end product carboxyethylpyrrole (CEP), which forms an adduct with fibrinogen and albumin via ε-amino group of lysines. Moreover, we revealed that macrophages adhered to CEP-modified albumin in α D β 2 -dependent manner. Now we are testing a hypothesis that DHA oxidation is a universal mechanism during chronic inflammatory diseases that promotes the generation of CEP adducts with different ECM proteins and forms α D β 2 -mediated strong anchorage of macrophages, which is critical for macrophage retention during chronic inflammation. We detected CEP-modified proteins in digested atherosclerotic lesions by western blot. In vitro DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin but not with collagen I. Using α D β 2 -transfected HEK293 cells, WT and α D -/- mouse macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading to compare with natural macrophage ligands. Using site-directed mutagenesis, we generated mutant α D I-domains and α D β 2 -transfected cells with single amino acid substitutions. Applying protein-protein binding and adhesion assays we detected one amino acid within integrin α D , K 246 , which is critical for α D β 2 binding to CEP-modified proteins. In summary, we propose a new mechanism of macrophage retention, which is based on inflammatory modifications of ECM with DHA end-product, CEP. The identification of a binding site for CEP-modified proteins within α D β 2 will help to develop a blocking reagent for the treatment of the inflammatory component of atherosclerosis.