Aging and cancer.

Aging and cancer exhibit apparent links that we will examine in this review. The null hypothesis that aging and cancer coincide because both are driven by time, irrespective of the precise causes, can be confronted with the idea that aging and cancer share common mechanistic grounds that are referred to as 'hallmarks'. Indeed, several hallmarks of aging also contribute to carcinogenesis and tumor progression, but some of the molecular and cellular characteristics of aging may also reduce the probability of developing lethal cancer, perhaps explaining why very old age (> 90 years) is accompanied by a reduced incidence of neoplastic diseases. We will also discuss the possibility that the aging process itself causes cancer, meaning that the time-dependent degradation of cellular and supracellular functions that accompanies aging produces cancer as a byproduct or 'age-associated disease'. Conversely, cancer and its treatment may erode health and drive the aging process, as this has dramatically been documented for cancer survivors diagnosed during childhood, adolescence, and young adulthood. We conclude that aging and cancer are connected by common superior causes including endogenous and lifestyle factors, as well as by a bidirectional crosstalk, that together render old age not only a risk factor of cancer but also an important parameter that must be considered for therapeutic decisions.

Inhibition of acyl-CoA binding protein (ACBP) by means of a GABAARγ2-derived peptide.

Acyl-CoA binding protein (ACBP) encoded by diazepam binding inhibitor (DBI) is an extracellular inhibitor of autophagy acting on the gamma-aminobutyric acid A receptor (GABAAR) γ2 subunit (GABAARγ2). Here, we show that lipoanabolic diets cause an upregulation of GABAARγ2 protein in liver hepatocytes but not in other major organs. ACBP/DBI inhibition by systemically injected antibodies has been demonstrated to mediate anorexigenic and organ-protective, autophagy-dependent effects. Here, we set out to develop a new strategy for developing ACBP/DBI antagonists. For this, we built a molecular model of the interaction of ACBP/DBI with peptides derived from GABAARγ2. We then validated the interaction between recombinant and native ACBP/DBI protein and a GABAARγ2-derived eicosapeptide (but not its F77I mutant) by pull down experiments or surface plasmon resonance. The GABAARγ2-derived eicosapeptide inhibited the metabolic activation of hepatocytes by recombinant ACBP/DBI protein in vitro. Moreover, the GABAARγ2-derived eicosapeptide (but not its F77I-mutated control) blocked appetite stimulation by recombinant ACBP/DBI in vivo, induced autophagy in the liver, and protected mice against the hepatotoxin concanavalin A. We conclude that peptidomimetics disrupting the interaction between ACBP/DBI and GABAARγ2 might be used as ACBP/DBI antagonists. This strategy might lead to the future development of clinically relevant small molecules of the ACBP/DBI system.

Orthotopic Model of Hepatocellular Carcinoma in Mice.

Orthotopic models of hepatocellular carcinoma (HCC) consist in the implantation of tumor cells into the liver by direct intrahepatic injection. In this model, tumorigenesis is triggered within the hepatic microenvironment, thus mimicking the metastatic behavior of HCC. Herein, we detail a surgically mediated methodology that allows the reproducible and effective induction of liver-sessile tumors in mice. We enumerate the steps to be followed before and after the surgical procedure, including HCC cell preparation, the quantity of cancer cells to be injected, presurgical preparation of the mice, and finally, postoperative care. The surgical procedure involves laparotomy to expose the liver, injection of cells into the left-lateral hepatic lobe, and closure of the incision with sutures followed by wound clips. We also provide information concerning the subsequent tumor growth follow-up, as well as the application of bioluminescence imaging to monitor tumor development.

A Mouse Model of Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma Induced by Western Diet and Carbon Tetrachloride.

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD). Obesity is a known risk factor of NASH, which, in turn, increases the risk of developing cirrhosis (liver scarring) and hepatocellular carcinoma (HCC). In addition to being a potentially life-threatening condition, public health concerns surrounding NASH are amplified by the lack of FDA-approved treatments. Although various preclinical models reflecting both the histopathology and the pathophysiological progression of human NASH exist, most of these models are diet-based and require 6-13 months for NASH symptom manifestation. Here, we describe a simple and rapid-progression model of NASH and NASH-driven HCC in mice. Mice received a western diet equivalent (WD; i.e., a high-fat, high-fructose, and high-cholesterol diet), high-sugar water (23.1 g/L fructose and 18.9 g/L glucose), and weekly intraperitoneal injections of carbon tetrachloride (CCl4) at a dose of 0.2 µL/g of body weight. The resulting phenotype, consisting in liver fibrosis and HCC, appeared within 24 weeks of diet/treatment initiation and presented similar histological and transcriptomic features as human NASH and NASH-driven HCC, thereby supporting the adequacy of this preclinical model for the development and evaluation of drugs that can prevent or reverse these diseases.

A Mouse Model of Hepatocellular Carcinoma Induced by Streptozotocin and High-Fat Diet.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the second most common cause of cancer-related death. HCC is associated to chronic diseases such as viral hepatitis, alcoholic, and non-alcoholic fatty liver disease (NAFLD), diabetes mellitus, and obesity, among others. Although pre-clinical models have been investigated to mimic the transition from NAFLD to HCC, they do not accurately reproduce the phenotypic evolution from simple steatosis to steatohepatitis, fibrosis/cirrhosis, and HCC. Hence, these models have failed to demonstrate the influence of diabetes on hepatic carcinogenesis. Here, we report a novel mouse model of HCC triggered by fast-developing diabetes and NAFLD. The first step consists in a single intraperitoneal injection of a low dose of streptozotocin into neonatal C57BL/6J mice to induce type 2 diabetes. In a second step, mice are fed with high-fat diet to accelerate the development of simple steatosis. Continuous high-fat diet exacerbates hepatic fat deposition with increased lobular inflammation (by activation of foam cell-like macrophages) and fibrosis (by activating hepatic stellate cells), two representative pathological traits of steatohepatitis/fibrosis. After 20 weeks, all mice developed multiple HCCs. This model of hepatic carcinogenesis triggered by diabetes mellitus and NAFLD offers the advantage of being rapid and accurately recapitulates the pathogenesis of human HCC without the need of administering hepatic carcinogens.

Targeted Analysis of Glycerophospholipids and Mono-, Di-, or Tri-Acylglycerides in Liver Cancer.

The metabolic rearrangements of hepatic metabolism associated with liver cancer are still incompletely understood. There is an ongoing need to identify novel and more efficient diagnostic biomarkers and therapeutic targets based on the metabolic mechanisms of these diseases. In comparison to traditional diagnostic biomarkers, metabolomics is a comprehensive technique for discovering chemical signatures for liver cancer screening, prediction, and earlier diagnosis. Lipids are a large and diverse group of complex biomolecules that are at the heart of liver physiology and play an important role in the development and progression of cancer. In this chapter, we described two detailed protocols for targeted lipids analysis: glycerophospholipids and mono, di, tri-acylglycerides, both by Flow Injection Analysis (FIA) HPLC coupled to a SelexIon/QTRAP 6500+ system. These approaches provide a targeted lipidomic metabolomic signature of dissimilar metabolic disorders affecting liver cancers.

Biomarker Identification in Liver Cancers Using Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) Imaging: An Approach for Spatially Resolved Metabolomics.

Liver cancers are characterized by interindividual and intratumoral heterogeneity, which makes early diagnosis and the development of therapies challenging. Desorption electrospray ionization mass spectrometry (DESI-MS) imaging is a potent and sensitive MS ionization technique for direct, unaltered 2D and 3D imaging of metabolites in complex biological samples. Indeed, DESI gently desorbs and ionizes analyte molecules from the sample surface using an electrospray source of highly charged aqueous spray droplets in ambient conditions. DESI-MS imaging of biological samples allows untargeted analysis and characterization of metabolites in liver cancers to identify new biomarkers of malignancy. In this chapter, we described a detailed protocol using liver cancer samples collected and stored for histopathology examination, either as frozen or as formalin-fixed, paraffin-embedded specimens. Such hepatocellular carcinoma samples can be subjected to DESI-MS analyses, illustrating the capacity of spatially resolved metabolomics to distinguish malignant lesions from adjacent normal liver tissue.

Formyl peptide receptor-1 (FPR1) represses intestinal oncogenesis.

Formyl peptide receptor-1 (FPR1) is a pattern recognition receptor that is mostly expressed by myeloid cells. In patients with colorectal cancer (CRC), a loss-of-function polymorphism (rs867228) in the gene coding for FPR1 has been associated with reduced responses to chemotherapy or chemoradiotherapy. Moreover, rs867228 is associated with accelerated esophageal and colorectal carcinogenesis. Here, we show that dendritic cells from Fpr1-/- mice exhibit reduced migration in response to chemotherapy-treated CRC cells. Moreover, Fpr1-/- mice are particularly susceptible to chronic ulcerative colitis and colorectal oncogenesis induced by the mutagen azoxymethane followed by oral dextran sodium sulfate, a detergent that induces colitis. These experiments were performed after initial co-housing of Fpr1-/- mice and wild-type controls, precluding major Fpr1-driven differences in the microbiota. Pharmacological inhibition of Fpr1 by cyclosporin H also tended to increase intestinal oncogenesis in mice bearing the ApcMin mutation, and this effect was reversed by the anti-inflammatory drug sulindac. We conclude that defective FPR1 signaling favors intestinal tumorigenesis through the modulation of the innate inflammatory/immune response.

Acyl coenzyme A binding protein (ACBP): An aging- and disease-relevant "autophagy checkpoint".

Acyl coenzyme A binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is a phylogenetically ancient protein present in some eubacteria and the entire eukaryotic radiation. In several eukaryotic phyla, ACBP/DBI transcends its intracellular function in fatty acid metabolism because it can be released into the extracellular space. This ACBP/DBI secretion usually occurs in response to nutrient scarcity through an autophagy-dependent pathway. ACBP/DBI and its peptide fragments then act on a range of distinct receptors that diverge among phyla, namely metabotropic G protein-coupled receptor in yeast (and likely in the mammalian central nervous system), a histidine receptor kinase in slime molds, and ionotropic gamma-aminobutyric acid (GABA)A receptors in mammals. Genetic or antibody-mediated inhibition of ACBP/DBI orthologs interferes with nutrient stress-induced adaptations such as sporulation or increased food intake in multiple species, as it enhances lifespan or healthspan in yeast, plant leaves, nematodes, and multiple mouse models. These lifespan and healthspan-extending effects of ACBP/DBI suppression are coupled to the induction of autophagy. Altogether, it appears that neutralization of extracellular ACBP/DBI results in "autophagy checkpoint inhibition" to unleash the anti-aging potential of autophagy. Of note, in humans, ACBP/DBI levels increase in various tissues, as well as in the plasma, in the context of aging, obesity, uncontrolled infection or cardiovascular, inflammatory, neurodegenerative, and malignant diseases.

DBI/ACBP is a targetable autophagy checkpoint involved in aging and cardiovascular disease.

DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) is a phylogenetically conserved paracrine inhibitor of macroautophagy/autophagy. As such, DBI/ACBP acts as a pro-aging molecule. Indeed, we observed that the knockout of ACB1 (the yeast equivalent of human DBI/ACBP) induces autophagy and prolongs lifespan in an autophagy-dependent fashion in chronological lifespan experiments. Intriguingly, circulating DBI/ACBP protein augments with age in humans, and this increase occurs independently from the known correlation of DBI/ACBP with body mass index (BMI). A supraphysiological DBI/ACBP level announces future cardiovascular disease (such as heart surgery, myocardial infarction and stroke) in still healthy individuals, suggesting that, beyond its correlation with chronological age, DBI/ACBP is a biomarker of biological age. Plasma DBI/ACBP concentrations correlate with triglycerides and anticorrelate with high-density lipoprotein. Of note, these associations with cardiovascular risk factors are independent from age and BMI in a multivariate regression model. In mice, we found that antibody-mediated neutralization of DBI/ACBP reduces signs of anthracycline-accelerated cardiac aging including the upregulation of the senescence marker CDKN2A/p16 (cyclin dependent kinase inhibitor 2A) and the functional decline of the heart. In conclusion, it appears that extracellular DBI/ACBP can be targeted to combat age-associated cardiovascular disease.Abbreviations: BMI: body mass index; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CVD: cardiovascular disease; DBI/ACBP: diazepam binding inhibitor, acyl-CoA binding protein; ELISA: enzyme-linked immunosorbent assay; GABA: gamma-aminobutyric acid; GABR: gamma-aminobutyric acid type A receptor.

High plasma concentrations of acyl-coenzyme A binding protein (ACBP) predispose to cardiovascular disease: Evidence for a phylogenetically conserved proaging function of ACBP.

Autophagy defects accelerate aging, while stimulation of autophagy decelerates aging. Acyl-coenzyme A binding protein (ACBP), which is encoded by a diazepam-binding inhibitor (DBI), acts as an extracellular feedback regulator of autophagy. As shown here, knockout of the gene coding for the yeast orthologue of ACBP/DBI (ACB1) improves chronological aging, and this effect is reversed by knockout of essential autophagy genes (ATG5, ATG7) but less so by knockout of an essential mitophagy gene (ATG32). In humans, ACBP/DBI levels independently correlate with body mass index (BMI) as well as with chronological age. In still-healthy individuals, we find that high ACBP/DBI levels correlate with future cardiovascular events (such as heart surgery, myocardial infarction, and stroke), an association that is independent of BMI and chronological age, suggesting that ACBP/DBI is indeed a biomarker of "biological" aging. Concurringly, ACBP/DBI plasma concentrations correlate with established cardiovascular risk factors (fasting glucose levels, systolic blood pressure, total free cholesterol, triglycerides), but are inversely correlated with atheroprotective high-density lipoprotein (HDL). In mice, neutralization of ACBP/DBI through a monoclonal antibody attenuates anthracycline-induced cardiotoxicity, which is a model of accelerated heart aging. In conclusion, plasma elevation of ACBP/DBI constitutes a novel biomarker of chronological aging and facets of biological aging with a prognostic value in cardiovascular disease.

ACBP/DBI protein neutralization confers autophagy-dependent organ protection through inhibition of cell loss, inflammation, and fibrosis.

Acyl-coenzyme A (CoA)-binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is an extracellular feedback regulator of autophagy. Here, we report that injection of a monoclonal antibody neutralizing ACBP/DBI (α-DBI) protects the murine liver against ischemia/reperfusion damage, intoxication by acetaminophen and concanavalin A, and nonalcoholic steatohepatitis caused by methionine/choline-deficient diet as well as against liver fibrosis induced by bile duct ligation or carbon tetrachloride. α-DBI downregulated proinflammatory and profibrotic genes and upregulated antioxidant defenses and fatty acid oxidation in the liver. The hepatoprotective effects of α-DBI were mimicked by the induction of ACBP/DBI-specific autoantibodies, an inducible Acbp/Dbi knockout or a constitutive Gabrg2F77I mutation that abolishes ACBP/DBI binding to the GABAA receptor. Liver-protective α-DBI effects were lost when autophagy was pharmacologically blocked or genetically inhibited by knockout of Atg4b. Of note, α-DBI also reduced myocardium infarction and lung fibrosis, supporting the contention that it mediates broad organ-protective effects against multiple insults.

Science-Driven Nutritional Interventions for the Prevention and Treatment of Cancer.

In population studies, dietary patterns clearly influence the development, progression, and therapeutic response of cancers. Nonetheless, interventional dietary trials have had relatively little impact on the prevention and treatment of malignant disease. Standardization of nutritional interventions combined with high-level mode-of-action studies holds the promise of identifying specific entities and pathways endowed with antineoplastic properties. Here, we critically review the effects of caloric restriction and more specific interventions on macro- and micronutrients in preclinical models as well as in clinical studies. We place special emphasis on the prospect of using defined nutrition-relevant molecules to enhance the efficacy of established anticancer treatments. SIGNIFICANCE: The avoidance of intrinsically hypercaloric and toxic diets contributes to the prevention and cure of cancer. In addition, specific diet-induced molecules such as ketone bodies and micronutrients, including specific vitamins, have drug-like effects that are clearly demonstrable in preclinical models, mostly in the context of immunotherapies. Multiple trials are underway to determine the clinical utility of such molecules.

An obesogenic feedforward loop involving PPARγ, acyl-CoA binding protein and GABAA receptor.

Acyl-coenzyme-A-binding protein (ACBP), also known as a diazepam-binding inhibitor (DBI), is a potent stimulator of appetite and lipogenesis. Bioinformatic analyses combined with systematic screens revealed that peroxisome proliferator-activated receptor gamma (PPARγ) is the transcription factor that best explains the ACBP/DBI upregulation in metabolically active organs including the liver and adipose tissue. The PPARγ agonist rosiglitazone-induced ACBP/DBI upregulation, as well as weight gain, that could be prevented by knockout of Acbp/Dbi in mice. Moreover, liver-specific knockdown of Pparg prevented the high-fat diet (HFD)-induced upregulation of circulating ACBP/DBI levels and reduced body weight gain. Conversely, knockout of Acbp/Dbi prevented the HFD-induced upregulation of PPARγ. Notably, a single amino acid substitution (F77I) in the γ2 subunit of gamma-aminobutyric acid A receptor (GABAAR), which abolishes ACBP/DBI binding to this receptor, prevented the HFD-induced weight gain, as well as the HFD-induced upregulation of ACBP/DBI, GABAAR γ2, and PPARγ. Based on these results, we postulate the existence of an obesogenic feedforward loop relying on ACBP/DBI, GABAAR, and PPARγ. Interruption of this vicious cycle, at any level, indistinguishably mitigates HFD-induced weight gain, hepatosteatosis, and hyperglycemia.

Improved Swiss-rolling method for histological analyses of colon tissue.

Since the introduction of the Swiss-rolling technique by Reilly and Kirsner in 1965, various methodological approaches have been developed for histological analyses of intestinal tissues. Here, we describe an improved protocol for the processing of freshly harvested murine colons that can be extended to other intestinal tissues. With simple tools, this technique allows to tightly wrap the organ throughout the whole length and to keep it in place before fixation, avoiding excessive stiffness of the tissue. Unlike the original method which relies on frozen samples, processing of the biological samples right after resection preserves epitopes integrity for subsequent immunohistochemical analyses. Ultimately, this method provides a reproducible workflow to capture the entire colon length in a unique histological section in order to assess several features such as intestinal inflammation and tumorigenesis. • Easily include freshly isolated tissues • Shorten preparation time using a few affordable tools • Prevent unrolling and preserve tissue integrity.

Immunization of mice with the self-peptide ACBP coupled to keyhole limpet hemocyanin.

Keyhole limpet hemocyanin (KLH) is a glycosylated multi-subunit metalloprotein that elicits a strong nonspecific immune activation, thus inducing both cellular and humoral immune responses. The exceptional immunogenicity of this protein can be leveraged to vaccinate mice against self-antigens that otherwise would not induce an autoimmune response. This protocol describes the covalent conjugation of KLH with acyl-coenzyme A-binding protein (ACBP), the autovaccination of mice with ACBP-KLH conjugate together with a potent adjuvant, and the detection of the produced anti-ACBP autoantibodies. For complete details on the use and execution of this profile, please refer to Bravo-San Pedro et al. (2019c).

Immunoblot-based assays for assessing autophagy in the turquoise killifish Nothobranchius furzeri.

Autophagy is an evolutionarily conserved biological process required for the turnover of the cytoplasm of eukaryotic cell. Beyond its catabolic nature, autophagy has a plethora of pro-survival functions, thus combatting hypoxia, nutrient shortage, and unfolded protein accumulation. Here, we introduce the naturally short-lived turquoise killifish Nothobranchius furzeri as an emerging model to study autophagic function in vivo, in response to environmental challenges. We show that starvation in killifish is sufficient to increase autophagic flux in the liver, thus enhancing the lipidation of microtubule-associated protein light chain 3 (LC3) and reducing the abundance of the autophagic substrate sequestosome-1 (SQSTM1). We describe an immunoblot-based comprehensive protocol to monitor fluctuations in autophagy in this model organism.

Multiplexed quantification of autophagic flux by imaging flow cytometry.

Imaging flow cytometry allows for the quantitative assessment of fluorescent signals at the subcellular level. Here, we describe the use of a biosensor cell line, namely, U2OS osteosarcoma cells equipped with a fusion protein comprising monomeric red fluorescent protein (mRFP), green fluorescent protein (GFP) and microtubule-associated proteins 1A/1B light chain 3B (best known as LC3), for the assessment of autophagic flux by imaging flow cytometry. We detail all analysis tools required to distinguish autophagosomes (that emit both a red and a green fluorescence) and autolysosomes (that emit a red fluorescence, yet lose the green fluorescent signal) and to quantitate autophagic flux in a convenient fashion.