SnapShot: FABP Functions.

Fatty acid binding proteins (FABPs) serve as intracellular chaperones for fatty acids and other hydrophobic ligands inside cells. Recent studies have demonstrated new functions of individual members of the FABP family. This Snapshot describes the overall functions of FABPs in health and disease and highlights emerging roles of adipose FABP (A-FABP) and epidermal FABP (E-FABP) in the fields of obesity, chronic inflammation, and cancer development. To view this SnapShot, open or download the PDF.

Development of a humanized anti-FABP4 monoclonal antibody for potential treatment of breast cancer.

BACKGROUND: Breast cancer is the most common cancer in women diagnosed in the U.S. and worldwide. Obesity increases breast cancer risk without clear underlying molecular mechanisms. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP, or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk, thus potentially offering a new target for breast cancer treatment. METHODS: We immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration, invasion, and limiting dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice, Balb/c mice, and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity, we determined the dissociation constant of selected anti-FABP4 antibodies via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. RESULTS: Herein, we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone, named 12G2, which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth, was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions, 16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. CONCLUSIONS: Our current evidence suggests that targeting FABP4 with humanized monoclonal antibodies may represent a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases.

Stearic Acid Induces CD11c Expression in Proinflammatory Macrophages via Epidermal Fatty Acid Binding Protein.

Obesity is associated with elevated levels of free fatty acids (FAs) and proinflammatory CD11c+ macrophages. However, whether and how free FAs contribute to CD11c+ macrophage differentiation and proinflammatory functions remain unclear. Here we report that dietary saturated FAs, but not unsaturated FAs, promoted the differentiation and function of CD11c+ macrophages. Specifically, we demonstrated that stearic acid (SA) significantly induced CD11c expression in monocytes through activation of the nuclear retinoid acid receptor. More importantly, cytosolic expression of epidermal FA binding protein (E-FABP) in monocytes/macrophages was shown to be critical to the mediation of the SA-induced effect. Depletion of E-FABP not only inhibited SA-induced CD11c upregulation in macrophages in vitro but also abrogated high-saturated-fat diet-induced skin lesions in obese mouse models in vivo. Altogether, our data demonstrate a novel mechanism by which saturated FAs promote obesity-associated inflammation through inducing E-FABP/retinoid acid receptor-mediated differentiation of CD11c+ macrophages.

Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-Induced Macrophage Cell Death through Enhancing Ceramide Production.

Macrophages play a critical role in obesity-associated chronic inflammation and disorders. However, the molecular mechanisms underlying the response of macrophages to elevated fatty acids (FAs) and their contribution to metabolic inflammation in obesity remain to be fully elucidated. In this article, we report a new mechanism by which dietary FAs, in particular, saturated FAs (sFAs), are able to directly trigger macrophage cell death. We demonstrated that excess sFAs, but not unsaturated FAs, induced the production of cytotoxic ceramides (Cers) in macrophage cell lines. Most importantly, expression of adipose FA binding protein (A-FABP) in macrophages facilitated metabolism of excess sFAs for Cer synthesis. Inhibition or deficiency of A-FABP in macrophage cell lines decreased sFA-induced Cer production, thereby resulting in reduced cell death. Furthermore, we validated the role of A-FABP in promoting sFA-induced macrophage cell death with primary bone marrow-derived macrophages and high-fat diet-induced obese mice. Altogether, our data reveal that excess dietary sFAs may serve as direct triggers in induction of Cer production and macrophage cell death through elevated expression of A-FABP, thus establishing A-FABP as a new molecular sensor in triggering macrophage-associated sterile inflammation in obesity.

Cancer-testis antigen HCA587/MAGE-C2 interacts with BS69 and promotes its degradation in the ubiquitin-proteasome pathway.

HCA587, also known as MAGE-C2, belonging to the MAGE gene family which is characterized by a conserved MAGE Homology Domain, is active in various types of tumors and silent in normal tissues except in male germ-line cells. The biological function of HCA587 is largely unknown. To analyze it, we attempted to identify protein partners of HCA587. We immunopurified HCA587-containing complex from HEK293 cells and identified BS69, a potential tumor suppressor, as an associated protein by mass spectrometry, and the following Immunoprecipitation and GST pull-down assays confirmed HCA587 interaction with BS69. Interestingly, overexpression of HCA587 promoted ubiquitination and the proteasomal degradation of BS69 whereas knockdown of endogenous HCA587 increased the protein level of BS69. Consistent with a functional role for BS69 in negatively regulating LMP1-induced NF-κB activation, overexpression of HCA587 resulted in a significant enhancement of LMP1-induced IL-6 production. These data indicate that HCA587 is a new negative regulator of BS69.

Determination of the FABP5 expression profile in skin lesions of an IMQ-induced psoriasis mouse model using flow cytometry.

The fatty acid-binding protein 5 (FABP5) is a key player in psoriasis development. Therefore, characterizing the expression profile of FABP5 in various cell types within both layers of psoriatic skin is important. Here, we present a protocol that describes steps for an imiquimod-induced psoriasis mouse model and preparation of epidermal and dermal single-cell suspensions. We then detail procedures to detect the FABP5 expression profile in skin keratinocytes and immune cells using intracellular flow cytometry staining. For complete details on the use and execution of this protocol, please refer to Hao et al.1.

FABP4-mediated lipid accumulation and lipolysis in tumor associated macrophages promote breast cancer metastasis.

A high density of tumor-associated macrophages (TAMs) is associated with poorer prognosis and survival in breast cancer patients. Recent studies have shown that lipid accumulation in TAMs can promote tumor growth and metastasis in various models. However, the specific molecular mechanisms that drive lipid accumulation and tumor progression in TAMs remain largely unknown. Herein, we demonstrated that unsaturated fatty acids (FAs), unlike saturated ones, are more likely to form lipid droplets in macrophages. Specifically, unsaturated FAs, including linoleic acids (LA), activate the FABP4/CEBPα pathway, leading to triglyceride synthesis and lipid droplet formation. Furthermore, FABP4 enhances lipolysis and FA utilization by breast cancer cells, which promotes cancer cell migration in vitro and metastasis in vivo . Notably, a deficiency of FABP4 in macrophages significantly reduces LA-induced lipid metabolism. Therefore, our findings suggest FABP4 as a crucial lipid messenger that facilitates unsaturated FA-mediated lipid accumulation and lipolysis in TAMs, thus contributing to the metastasis of breast cancer. Highlights: Unlike saturated fatty acids, unsaturated fatty acids preferentially promote lipid droplet formation in macrophages.Unsaturated fatty acids activate the FABP4/CEBPα axis for neutral lipid biosynthesis in macrophagesDeficiency of FABP4 compromised unsaturated fatty acid-mediated lipid accumulation and utilization in macrophagesFABP4-mediated lipid metabolism in macrophages contributes to breast cancer metastasis.

Role of FABP5 in T Cell Lipid Metabolism and Function in the Tumor Microenvironment.

To evade immune surveillance, tumors develop a hostile microenvironment that inhibits anti-tumor immunity. Recent immunotherapy breakthroughs that target the reinvigoration of tumor-infiltrating T lymphocytes (TIL) have led to unprecedented success in treating some cancers that are resistant to conventional therapy, suggesting that T cells play a pivotal role in anti-tumor immunity. In the hostile tumor microenvironment (TME), activated T cells are known to mainly rely on aerobic glycolysis to facilitate their proliferation and anti-tumor function. However, TILs usually exhibit an exhausted phenotype and impaired anti-tumor activity due to the limited availability of key nutrients (e.g., glucose) in the TME. Given that different T cell subsets have unique metabolic pathways which determine their effector function, this review introduces our current understanding of T cell development, activation signals and metabolic pathways. Moreover, emerging evidence suggests that fatty acid binding protein 5 (FABP5) expression in T cells regulates T cell lipid metabolism and function. We highlight how FABP5 regulates fatty acid uptake and oxidation, thus shaping the survival and function of different T cell subsets in the TME.

Keratinocyte FABP5-VCP complex mediates recruitment of neutrophils in psoriasis.

One of the hallmarks of intractable psoriasis is neutrophil infiltration in skin lesions. However, detailed molecular mechanisms of neutrophil chemotaxis and activation remain unclear. Here, we demonstrate a significant upregulation of epidermal fatty acid binding protein (E-FABP, FABP5) in the skin of human psoriasis and psoriatic mouse models. Genetic deletion of FABP5 in mice by global knockout and keratinocyte conditional (Krt6a-Cre) knockout, but not myeloid cell conditional (LysM-Cre) knockout, attenuates psoriatic symptoms. Immunophenotypic analysis shows that FABP5 deficiency specifically reduces skin recruitment of Ly6G+ neutrophils. Mechanistically, activated keratinocytes produce chemokines and cytokines that trigger neutrophil chemotaxis and activation in an FABP5-dependent manner. Proteomic analysis further identifies that FABP5 interacts with valosin-containing protein (VCP), a key player in NF-κB signaling activation. Silencing of FABP5, VCP, or both inhibits NF-κB/neutrophil chemotaxis signaling. Collectively, these data demonstrate dysregulated FABP5 as a molecular mechanism promoting NF-κB signaling and neutrophil infiltration in psoriasis pathogenesis.

Epidermal Fatty Acid‒Binding Protein Mediates Depilatory-Induced Acute Skin Inflammation.

Depilatory creams are widely used to remove unwanted body hair, but people with sensitive skin are subject to depilatory-induced skin burn/inflammation. It remains unknown what makes their skin more sensitive than others. In this study, we show that epidermal fatty acid‒binding protein (E-FABP) expressed in the skin plays a critical role in promoting depilatory-induced acute skin inflammation in mouse models. Although a depilatory cream removed hair by breaking down keratin disulfide bonds, it activated cytosolic phospholipase A2, leading to activation of the arachidonic acid/E-FABP/peroxisome proliferator-activated receptor ß signaling pathway in keratinocytes. Specifically, peroxisome proliferator-activated receptor ß activation induced downstream targets (e.g., cyclooxygenase 2) and chemokine (e.g., CXCL1) production, which systemically mobilized neutrophils and recruited them to localize in the skin for acute inflammatory responses. Importantly, E-FABP deletion by CRISPR-Cas9 reduced cytosolic phospholipase A2/peroxisome proliferator-activated receptor ß activation in keratinocytes, and genetic deletion of E-FABP protected mice from depilatory cream-induced neutrophil recruitment and skin inflammation. Our findings suggest E-FABP as a molecular sensor for sensitive skin by triggering depilatory-induced, lipid-mediated skin inflammatory responses.

Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages.

Fats are essential in healthy diets, but how dietary fats affect immune cell function and overall health is not well understood. Mimicking human high-fat diets (HFDs), which are rich in different fatty acid (FA) components, we fed mice various HFDs from different fat sources, including fish oil and cocoa butter. Mice consuming the fish oil HFD exhibit a hair-loss phenotype. Further studies show that omega-3 (n-3) FAs in fish oil promote atypical infiltration of CD207- (langerin-) myeloid macrophages in skin dermis, which induce hair loss through elevated TNF-α signaling. Mechanistically, epidermal fatty acid binding protein (E-FABP) is demonstrated to play an essential role in inducing TNF-α-mediated hair loss by activating the n-3 FA/ROS/IL-36 signaling pathway in dermal resident macrophages. Absence of E-FABP abrogates fish oil HFD-induced murine hair loss. Altogether, these findings support a role for E-FABP as a lipid sensor mediating n-3 FA-regulated macrophage function and skin health.

Identification of promiscuous HLA-DR-restricted CD4⁺ T-cell epitopes on the cancer-testis antigen HCA587.

The cancer testis antigen HCA587 is an attractive candidate for T cell-based immunotherapy because it is overexpressed in a wide spectrum of malignant tumors but not normal tissues, except testis. Several CTL epitopes derived from HCA587 have been described. Our aim was to identify helper T lymphocyte epitopes of HCA587 for the optimization of T cell-based immunotherapies against HCA587-expressing tumors. Candidate helper T lymphocyte epitopes for HCA587 were predicted using the SYFPEITHI algorithm and were tested for their ability to induce helper T lymphocyte responses by in vitro peptide vaccination of CD4(+) T lymphocytes from healthy individuals and hepatocellular carcinoma patients. Four CD4(+) T-cell epitopes for HCA587 (p43-57, p145-159, p186-200 and p249-263) were identified. Among them, the p43-57 epitope was shown to be naturally processed and presented by HCA587-expressing tumor cells as well as autologous dendritic cells pulsed with whole-protein HCA587. Notably, this epitope behaved as a promiscuous T-cell epitope as it stimulated T cells in the context of more than one HLA class II allele. Thus, p43-57 is the first HCA587-derived major histocompatibility complex class II-restricted epitope to fulfil all prerequisites for use as a peptide vaccine in patients with HCA587-expressing tumors.

Thermal Shift Assay for Exploring Interactions Between Fatty Acid-Binding Protein and Inhibitors.

Thermal shift assay (TSA) is a widely used method in discovering potential compounds (e.g., ligands, inhibitors, and other additives) to the target protein for structural genomics and drug screening in both academia and industry. The presence of sensitive fluorescent dye enables to monitor thermal stability of protein and compounds affecting this stability. By using a conventional real-time PCR instrument, it is determined as a low-cost and high efficacy experiment applied to identify optimal conditions for ligand binds to protein. Fatty acid-binding proteins (FABPs) are small molecular proteins in transporting fatty acids and other lipophilic substances in physiological and pathological responses. This chapter presents a comprehensive workflow to monitor recombinant FABP-compound interactions for an initial screening for inhibitors using TSA with SYPRO Orange dye.

Effects of interactions in natural gas/water/rock system on hydrocarbon migration and accumulation.

The study of natural gas accumulation process in tight formation has become the focus of the petroleum industry. One of the priorities is the effects of interactions in natural gas/water/rock system on hydrocarbon migration and accumulation process. On the macroscopic scale, we investigate the interactions in natural gas/water/rock system by formation fluorescence test and production data analysis. One the microscopic scale, the mechanisms are revealed by mathematical analysis and experimental methods considering the variation of geological temperature and pressure. The effects of interactions in natural gas/water/rock system are also simulated by numerical simulation. The results are visualized and quantified. A novel semi-analytical method based on a physical experiment is proposed to calculate the temperature- and pressure-dependent contact angle and interface tension which reflect the interactions in the natural gas-water-rock system. This semi-analytical is embedded in the numerical simulation during the simulation of the natural gas charging process. The results indicate that with the increase of geological temperature and pressure, the contact angle will increase and the interface tension between natural gas and water will decrease. The capillary resistance in the formation will be reduced. Since the decrease of capillary resistance, the natural gas can be charged into smaller pores, so that the actual charging threshold is lower than the one originally obtained under present reservoir conditions. After considering the temperature and pressure during the accumulation process, some sand bodies that were thought not to be charged may have natural gas accumulate.

Regulation of macrophage functions by FABP-mediated inflammatory and metabolic pathways.

Macrophages are almost everywhere in the body, where they serve pivotal functions in maintaining tissue homeostasis, remodeling, and immunoregulation. Macrophages are traditionally thought to differentiate from bone marrow-derived hematopoietic stem cells (HSCs). Emerging studies suggest that some tissue macrophages at steady state originate from embryonic precursors in the yolk sac or fetal liver and are maintained in situ by self-renewal, but bone marrow-derived monocytes can give rise to tissue macrophages in pathogenic settings, such as inflammatory injuries and cancer. Macrophages are popularly classified as Th1 cytokine (e.g. IFNγ)-activated M1 macrophages (the classical activation) or Th2 cytokine (e.g. IL-4)-activated M2 macrophages (the alternative activation). However, given the myriad arrays of stimuli macrophages may encounter from local environment, macrophages exhibit notorious heterogeneity in their phenotypes and functions. Determining the underlying metabolic pathways engaged during macrophage activation is critical for understanding macrophage phenotypic and functional adaptivity under different disease settings. Fatty acid binding proteins (FABPs) represent a family of evolutionarily conserved proteins facilitating lipid transport, metabolism and responses inside cells. More specifically, adipose-FABP (A-FABP) and epidermal-FABP (E-FABP) are highly expressed in macrophages and play a central role in integrating metabolic and inflammatory pathways. In this review we highlight how A-FABP and E-FABP are respectively upregulated in different subsets of activated macrophages and provide a unique perspective in defining macrophage phenotypic and functional heterogeneity through FABP-regulated lipid metabolic and inflammatory pathways.

Dietary Fats High in Linoleic Acids Impair Antitumor T-cell Responses by Inducing E-FABP-Mediated Mitochondrial Dysfunction.

The most recent American Dietary Guidelines (2020-2025) recommend shifting dietary fats from solid saturated fats to unsaturated oils. Dietary oils contain different compositions of unsaturated fatty acids (UFA). Oleic acid (OA) and linoleic acid (LA) are the most common UFA in dietary oils. How individual UFA in oils regulate immune cell function and cancer risk remains unclear. Here we demonstrated that high-fat diets (HFD) rich either in OA or LA induced a similar degree of murine obesity, but the LA-rich HFD specifically promoted mammary tumor growth. LA impaired antitumor T-cell responses by promoting naïve T-cell apoptosis and inhibiting TNFα production. While exogenous OA and LA were taken up by T cells with similar efficacy, only LA induced significant mitochondrial reactive oxygen species production and lipid peroxidation. Importantly, naïve T cells predominantly expressed epidermal fatty acid binding protein (E-FABP), which is central in facilitating LA mitochondrial transport and cardiolipin incorporation. Genetic depletion of E-FABP rescued LA-impaired T-cell responses and suppressed LA-rich HFD-associated mammary tumor growth. Collectively, these data suggest that dietary oils high in LA promote mammary tumors by inducing E-FABP-mediated T-cell dysfunction. SIGNIFICANCE: These findings suggest that modulation of dietary oil composition and inhibition of E-FABP activity may represent novel strategies to enhance T-cell function in the prevention and treatment of obesity-associated cancers.

Consumption of the Fish Oil High-Fat Diet Uncouples Obesity and Mammary Tumor Growth through Induction of Reactive Oxygen Species in Protumor Macrophages.

Obesity is associated with increased risk of many types of cancer and can be induced by various high-fat diets (HFD) from different fat sources. It remains unknown whether fatty acid composition in different HFD influences obesity-associated tumor development. Here we report that consumption of either a cocoa butter or fish oil HFD induced similar obesity in mouse models. While obesity induced by the cocoa butter HFD was associated with accelerated mammary tumor growth, consumption of the fish oil HFD uncoupled obesity from increased mammary tumor growth and exhibited a decrease in protumor macrophages. Compared with fatty acid (FA) components in both HFDs, n-3 FA rich in the fish oil HFD induced significant production of reactive oxygen species (ROS) and macrophage death. Moreover, A-FABP expression in the protumor macrophages facilitated intracellular transportation of n-3 FA and oxidation of mitochondrial FA. A-FABP deficiency diminished n-3 FA-mediated ROS production and macrophage death in vitro and in vivo. Together, our results demonstrate a novel mechanism by which n-3 FA induce ROS-mediated protumor macrophage death in an A-FABP-dependent manner. SIGNIFICANCE: This study provides mechanistic insight into dietary supplementation with fish oil for breast cancer prevention and advances a new concept that not all HFDs leading to obesity are tumorigenic. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/12/2564/F1.large.jpg.

A-FABP and oestrogens are independently involved in the development of breast cancer.

We previously reported that postmenopausal obese women exhibit increased levels of circulating adipocyte fatty acid binding protein (A-FABP), which is associated with breast cancer (BC) development. In postmenopause, increased oestrogen levels are reported to be associated with increased BC risk. Herein, we assessed if oestrogens, including oestrone (E1), oestradiol (E2) and oestriol (E3), are associated with A-FABP in the obesity-related BC development. We collected 249 serum samples from women with or without BC and measured serum levels of E1, E2, E3 and A-FABP. Considering all subjects, E1 and E2 but not E3 levels were significantly higher in pre- than in postmenopause individuals. E3 and E1 levels were higher in non-obese than in obese women. When samples were separated by BC status, E2 levels were significantly higher, while E1 and E3 levels were significantly lower in postmenopausal obese than non-obese women without BC. These differences based on body mass index (BMI) were not observed among women with BC. E3 levels were higher in obese women with BC than those without. A-FABP levels were significantly higher in postmenopausal obese women regardless of BC status. In addition, A-FABP was not associated with E1, E2 or E3. Altogether, our data suggest that A-FABP is independently regulated by obesity and menopausal status compared to oestrogens, thus playing a unique role in the development of BC.

Epidermal FABP Prevents Chemical-Induced Skin Tumorigenesis by Regulation of TPA-Induced IFN/p53/SOX2 Pathway in Keratinocytes.

Skin lipids (e.g., fatty acids) are essential for normal skin functions. Epidermal FABP (E-FABP) is the predominant FABP expressed in skin epidermis. However, the role of E-FABP in skin homeostasis and pathology remains largely unknown. Herein, we utilized the 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanolyphorbol-13-acetate-induced skin tumorigenesis model to assess the role of E-FABP in chemical-induced skin tumorigenesis. Compared to their wild-type littermates, mice deficient in E-FABP, but not adipose FABP, developed more skin tumors with higher incidence. 12-O-tetradecanolyphorbol-13-acetate functioning as a tumor promoter induced E-FABP expression and initiated extensive flaring inflammation in skin. Interestingly, 12-O-tetradecanolyphorbol-13-acetate -induced production of IFN-ß and IFN-λ in the skin tissue was dependent on E-FABP expression. Further protein and gene expression arrays demonstrated that E-FABP was critical in enhancing IFN-induced p53 responses and in suppressing SOX2 expression in keratinocytes. Thus, E-FABP expression in skin suppresses chemical-induced skin tumorigenesis through regulation of IFN/p53/SOX2 pathway. Collectively, our data suggest an unknown function of E-FABP in prevention of skin tumor development, and offer E-FABP as a therapeutic target for improving skin innate immunity in chemical-induced skin tumor prevention.

Circulating Adipose Fatty Acid Binding Protein Is a New Link Underlying Obesity-Associated Breast/Mammary Tumor Development.

It is clear that obesity increases the risk of many types of cancer, including breast cancer. However, the underlying molecular mechanisms by which obesity is linked to cancer risk remain to be defined. Herein, we report that circulating adipose fatty acid binding protein (A-FABP) promotes obesity-associated breast cancer development. Using clinical samples, we demonstrated that circulating A-FABP levels were significantly increased in obese patients with breast cancer in comparison with those without breast cancer. Circulating A-FABP released by adipose tissue directly targeted mammary tumor cells, enhancing tumor stemness and aggressiveness through activation of the IL-6/STAT3/ALDH1 pathway. Importantly, genetic deletion of A-FABP successfully reduced tumor ALHD1 activation and obesity-associated mammary tumor growth and development in different mouse models. Collectively, these data suggest circulating A-FABP as a new link between obesity and breast cancer risk, thereby revealing A-FABP as a potential new therapeutic target for treatment of obesity-associated cancers.