PCSK9 in Liver Cancers at the Crossroads between Lipid Metabolism and Immunity.

Metabolic rewiring and defective immune responses are considered to be the main driving forces sustaining cell growth and oncogenesis in many cancers. The atypical enzyme, proprotein convertase subtilisin/kexin type 9 (PCSK9), is produced by the liver in large amounts and plays a major role in lipid metabolism via the control of the low density lipoprotein receptor (LDLR) and other cell surface receptors. In this context, many clinical studies have clearly demonstrated the high efficacy of PCSK9 inhibitors in treating hyperlipidemia and cardiovascular diseases. Recent data implicated PCSK9 in the degradation of major histocompatibility complex I (MHC-I) receptors and the immune system as well as in other physiological activities. This review highlights the complex crosstalk between PCSK9, lipid metabolism and immunosuppression and underlines the latest advances in understanding the involvement of this convertase in other critical functions. We present a comprehensive assessment of the different strategies targeting PCSK9 and show how these approaches could be extended to future therapeutic options to treat cancers with a main focus on the liver.

Targeting PCSK9 in Liver Cancer Cells Triggers Metabolic Exhaustion and Cell Death by Ferroptosis.

Deregulated lipid metabolism is a common feature of liver cancers needed to sustain tumor cell growth and survival. We aim at taking advantage of this vulnerability and rewiring the oncogenic metabolic hub by targeting the key metabolic player pro-protein convertase subtilisin/kexin type 9 (PCSK9). We assessed the effect of PCSK9 inhibition using the three hepatoma cell lines Huh6, Huh7 and HepG2 and validated the results using the zebrafish in vivo model. PCSK9 deficiency led to strong inhibition of cell proliferation in all cell lines. At the lipid metabolic level, PCSK9 inhibition was translated by an increase in intracellular neutral lipids, phospholipids and polyunsaturated fatty acids as well as a higher accumulation of lipid hydroperoxide. Molecular signaling analysis involved the disruption of the sequestome 1/Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (p62/Keap1/Nrf2) antioxidative axis, leading to ferroptosis, for which morphological features were confirmed by electron and confocal microscopies. The anti-tumoral effects of PCSK9 deficiency were validated using xenograft experiments in zebrafish. The inhibition of PCSK9 was effective in disrupting the oncometabolic process, inducing metabolic exhaustion and enhancing the vulnerability of cancer cells to iron-triggered lipid peroxidation. We provide strong evidence supporting the drug repositioning of anti-PCSK9 approaches to treat liver cancers.

Rewiring Lipid Metabolism by Targeting PCSK9 and HMGCR to Treat Liver Cancer.

Alterations in lipid handling are an important hallmark in cancer. Our aim here is to target key metabolic enzymes to reshape the oncogenic lipid metabolism triggering irreversible cell breakdown. We targeted the key metabolic player proprotein convertase subtilisin/kexin type 9 (PCSK9) using a pharmacological inhibitor (R-IMPP) alone or in combination with 3-hydroxy 3-methylglutaryl-Coenzyme A reductase (HMGCR) inhibitor, simvastatin. We assessed the effect of these treatments using 3 hepatoma cell lines, Huh6, Huh7 and HepG2 and a tumor xenograft in chicken choriorallantoic membrane (CAM) model. PCSK9 deficiency led to dose-dependent inhibition of cell proliferation in all cell lines and a decrease in cell migration. Co-treatment with simvastatin presented synergetic anti-proliferative effects. At the metabolic level, mitochondrial respiration assays as well as the assessment of glucose and glutamine consumption showed higher metabolic adaptability and surge in the absence of PCSK9. Enhanced lipid uptake and biogenesis led to excessive accumulation of intracellular lipid droplets as revealed by electron microscopy and metabolic tracing. Using xenograft experiments in CAM model, we further demonstrated the effect of anti-PCSK9 treatment in reducing tumor aggressiveness. Targeting PCSK9 alone or in combination with statins deserves to be considered as a new therapeutic option in liver cancer clinical applications.

Immuno-Metabolic Modulation of Liver Oncogenesis by the Tryptophan Metabolism.

Metabolic rewiring in tumor cells is a major hallmark of oncogenesis. Some of the oncometabolites drive suppressive and tolerogenic signals from the immune system, which becomes complicit to the advent and the survival of neoplasia. Tryptophan (TRP) catabolism through the kynurenine (KYN) pathway was reported to play immunosuppressive actions across many types of cancer. Extensive debate of whether the culprit of immunosuppression was the depletion of TRP or rather KYN accumulation in the tumor microenvironment has been ongoing for years. Results from clinical trials assessing the benefit of inhibiting key limiting enzymes of this pathway such as indoleamine 2,3-dioxygenase (IDO1) or tryptophan 2,3-dioxygenase (TDO2) failed to meet the expectations. Bearing in mind the complexity of the tumoral terrain and the existence of different cancers with IDO1/TDO2 expressing and non-expressing tumoral cells, here we present a comprehensive analysis of the TRP global metabolic hub and the driving potential of the process of oncogenesis with the main focus on liver cancers.

Specific autoantigens in experimental autoimmunity-associated atherosclerosis.

Higher cardiovascular morbidity in patients with a wide range of autoimmune diseases highlights the importance of autoimmunity in promoting atherosclerosis. Our purpose was to investigate the mechanisms of accelerated atherosclerosis and identified vascular autoantigens targeted by autoimmunity. We created a mouse model of autoimmunity-associated atherosclerosis by transplanting bone marrow from FcγRIIB knockout (FcRIIB(-/-)) mice into LDL receptor knockout mice. We characterized the cellular and molecular mechanisms of atherogenesis and identified specific aortic autoantigens using serologic proteomic studies. En face lesion area analysis showed more aggressive atherosclerosis in autoimmune mice compared with control mice (0.64 ± 0.12 vs 0.32 ± 0.05 mm(2); P < 0.05, respectively). At the cellular level, FcRIIB(-/-) macrophages showed significant reduction (46-72%) in phagocytic capabilities. Proteomic analysis revealed circulating autoantibodies in autoimmune mice that targeted 25 atherosclerotic lesion proteins, including essential components of adhesion complex, cytoskeleton, and extracellular matrix, and proteins involved in critical functions and pathways. Microscopic examination of atherosclerotic plaques revealed essential colocalization of autoantibodies with endothelial cells, their adherence to basement membranes, the internal elastica lamina, and necrotic cores. The new vascular autoimmunosome may be a useful target for diagnostic and immunotherapeutic interventions in autoimmunity-associated diseases that have accelerated atherosclerosis.-Merched, A. J., Daret, D., Li, L., Franzl, N., Sauvage-Merched, M. Specific autoantigens in experimental autoimmunity-associated atherosclerosis.

Nutrigenetics and nutrigenomics of atherosclerosis.

The latest genome-wide association studies (GWAS) have re-energized our effort to understand the genetic basis of atherosclerotic cardiovascular disease. Although the knowledge generated by GWAS has confirmed that mediators of inflammation and perturbed lipid metabolism are major players in cardiovascular disease (CVD) development, much of individual disease heritability remains unexplained by the variants identified through GWAS. Moreover, results from interventions that aim at the pharmaceutical modification of lipid parameters fall short of expectation. These elusive treatment goals based on heritability studies highlight a key supportive, and perhaps even primary, role of nutritional therapy to achieve better health outcomes. Nonetheless, effective and specific interventions for CVD prevention using principles of "personalized" nutrition require a better knowledge of gene-diet interactions, an area that remains poorly explored. Dietary fatty acids such as omega-3 polyunsaturated fatty acids (PUFAs) are an excellent example of a widely studied "environment" that interacts with the genetic makeup in relation to CVD. A thorough exploration of the nutrigenomics and nutrigenetics of omega-3 PUFAs is key to understanding the etiology, and developing effective preventive measures. In this review, we will summarize the current state of knowledge of genetic interactions with omega-3 PUFAs in modulating lipid metabolism and inflammation, and defining health outcomes. Nutrigenetics and nutrigenomics are still in their infancy with respect to CVD prediction and therapy. Integration of the progress in the omics, including metabolomics, lipidomics, transcriptomics, and proteomics, coupled with advances in nutrigenomic and nutrigenetic research will move us towards personalized medicine as the ultimate paradigm of responsible clinical practice.

Nutrigenetic disruption of inflammation-resolution homeostasis and atherogenesis.

BACKGROUND/AIM: Pro-resolving and anti-inflammatory mediator products of murine 12/15-lipoxygenase (LOX) exhibit potent actions on vascular inflammation and protect against the progression of atherosclerosis. The present study was designed to determine whether augmenting dietary lipids modulates the body's endogenous anti-inflammatory pro-resolving mechanisms and promotes atherosclerosis. METHODS/RESULTS: We investigated the biometabolic consequences of variations in lipid mediator biosynthesis using genetic knockout and overexpression models of 12/15-LOX mice fed the commonly used 'Western diet'. Unexpectedly, this high-fat diet annulled the protective actions of 12/15-LOX, and the combination of a Western diet and 12/15-LOX overexpression paradoxically promoted inflammation leading to production of diet-related and 12/15-LOX-dependent blood mediators that differentially activated endothelial cells via expression of ICAM-1. Hyperlipidemia not only affected the biosynthesis of lipoxin A4, a key pro-resolving mediator, but also disrupted the protective pro-resolving function of 12/15-LOX products, and the enzyme pathway no longer protected against atherosclerosis in vivo. CONCLUSION: We uncovered a novel mechanism whereby a high-fat diet as well as hyperlipidemia disrupt the homeostasis of inflammation resolution. These findings underscore the importance of dietary essential PUFAs and LOX-derived lipid mediators in combination with lipid-lowering agents in the prevention and treatment of atherosclerotic cardiovascular diseases.

Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators.

Atherosclerosis is now recognized as an inflammatory disease involving the vascular wall. Recent results indicate that acute inflammation does not simply passively resolve as previously assumed but is actively terminated by a homeostatic process that is governed by specific lipid-derived mediators initiated by lipoxygenases. Experiments with animals and humans support a proinflammatory role for the 5-lipoxygenase system. In contrast, results from animal experiments show a range of responses with the 12/15-lipoxygenase pathways in atherosclerosis. To date, the only two clinical epidemiology human studies both support an antiatherogenic role for 12/15-lipoxygenase downstream actions. We tested the hypothesis that atherosclerosis results from a failure in the resolution of local inflammation by analyzing apolipoprotein E-deficient mice with 1) global leukocyte 12/15-lipoxygenase deficiency, 2) normal enzyme expression, or 3) macrophage-specific 12/15-lipoxygenase overexpression. Results from these indicate that 12/15-lipoxygenase expression protects mice against atherosclerosis via its role in the local biosynthesis of lipid mediators, including lipoxin A(4), resolvin D1, and protectin D1. These mediators exert potent agonist actions on macrophages and vascular endothelial cells that can control the magnitude of the local inflammatory response. Taken together, these findings suggest that a failure of local endogenous resolution mechanisms may underlie the unremitting inflammation that fuels atherosclerosis.

Nicotine induces proinflammatory responses in macrophages and the aorta leading to acceleration of atherosclerosis in low-density lipoprotein receptor(-/-) mice.

OBJECTIVE: We investigated the molecular mechanism of nicotine-accelerated atherosclerosis in a hyperlipidemic low-density lipoprotein receptor(-/-) mouse model. METHODS AND RESULTS: Low-density lipoprotein receptor(-/-) mice received time-release nicotine or placebo pellets for 90 days. Aortic lesion size was 2.5 times larger in nicotine-treated than in placebo-treated mice (P<0.001). A mild increase in lipids was seen in treated mice. We quantified 18 different serum cytokines and found a significant increase of tumor necrosis factor alpha, interleukin 1beta, and keratinocyte-derived chemokine in nicotine-treated mice. Among 107 nuclear factor kappaB (NF-kappaB) target genes screened from the aorta, we found that nicotine treatment upregulated only 4 atherogenic genes including vascular adhesion molecule 1 and cyclooxygenase 2 on day 60 and platelet-derived growth factor B and platelet 12-lipoxygenase on day 90. At the cellular level, nicotine induced tumor necrosis factor alpha and inducible nitric oxide synthase expression in RAW264.7 cells via the nicotinic acetylcholine receptors. Induction was confirmed in peritoneal macrophages isolated from nicotine-treated mice. Finally, we showed that preconditioned medium from nicotine-treated RAW264.7 cells activated NF-kappaB in human smooth muscle cells and vascular endothelial cells as evidenced by nuclear localization and electromobility shift assay. CONCLUSIONS: Chronic nicotine exposure augments atherosclerosis by enhancing the production of proinflammatory cytokines by macrophages, which, in turn, activate atherogenic NF-kappaB target genes in the aortic lesions.

Absence of p21Waf1/Cip1/Sdi1 modulates macrophage differentiation and inflammatory response and protects against atherosclerosis.

BACKGROUND: The tumor suppressor p53 protects against atherosclerosis progression in several different mouse models. A major target of p53 is p21, the cyclin-dependent kinase inhibitor that regulates entry into the cell cycle of different types of cells, including stem cells. p21 is also involved in the maturation and differentiation of monocytes into macrophages. METHODS AND RESULTS: We studied the effect of p21Waf1 inactivation on atherosclerosis development in apolipoprotein E-deficient mice (apoE-/-). Contrary to previous data suggesting a protective role for p21, we found that absence of p21, either globally or in bone marrow-derived cells, protects against atherosclerosis. Atherosclerotic lesions of p21-/-/apoE-/- mice exhibit a more stable phenotype, with increased apoptosis and reduced inflammatory vascular cell adhesion molecule-1 immunostaining but no difference in cellular proliferation compared with lesions of p21+/+/apoE-/- mice. Because bone marrow-derived cells mediate many of the effects of p21, we examined the expression profile of 23 genes in macrophages using real-time polymerase chain reaction. Compared with their p21+/+ counterparts, peritoneal macrophages of p21-/- mice express lower levels of proinflammatory markers, including macrophage inflammatory proteins 1 and 2 and interleukin-1alpha, and higher levels of putative protective genes, such as scavenger receptor type B-I and LDL receptor-related protein. Furthermore, we found that, in comparison with p21+/+ macrophages, p21-/- macrophages displayed increased phagocytic activity toward fluorescent latex microspheres as well as apoptotic cells, thus uncovering a novel mechanism of the antiinflammatory activity of p21-/- macrophages. CONCLUSIONS: Loss of p21 protects against atherosclerosis in apoE-/- mice. The data underscore the important role of p21 in macrophage function and inflammation and provide insight into the mechanism of the proatherogenic effect of p21.

Macrophage-specific p53 expression plays a crucial role in atherosclerosis development and plaque remodeling.

OBJECTIVE: We first showed that absence of p53 accelerates atherosclerosis development in apoE-deficient mice. In this study, we investigated how macrophage-specific loss of p53 function might modulate atherosclerosis development in LDL receptor-deficient mice, a model for familial hypercholesterolemia. METHODS AND RESULTS: We transferred bone marrow cells isolated from p53+/+ and p53-/- mice to lethally irradiated LDL receptor-/- mice and evaluated the aortic atherosclerotic lesion areas in the recipients at different times afterward. At 15 weeks and again at 20 weeks, we found larger aortic lesion size in mice receiving p53-/- cells compared with those that received p53+/+ cells. By measuring the rate of bromodeoxyuridine incorporation, we found that the absence of p53 in macrophages stimulates cellular proliferation. In contrast, the rate of apoptosis in the atheromatous lesion was similar in the two groups of mice. Furthermore, we found that the absence of macrophage-specific p53 expression was associated with vulnerable-appearing lesions marked by increased tissue necrosis and reduced collagen deposition. CONCLUSIONS: p53 plays a crucial role in atherosclerosis lesion development and remodeling, and macrophage-specific p53 deficiency stimulates cellular proliferation leading to a vulnerable-appearing phenotype of lesions in a mouse model of familial hypercholesterolemia.