REVIEW Mechanisms of metabolic dysfunction in cancer-associated cachexia Michele Petruzzelli1 and Erwin F. Wagner2 1Department of Oncology, The Medical Research Council Cancer Unit, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdon; 2Genes, Development, and Disease Group, Cancer Cell Biology Programme, Centro Nacional de Investigaciones Oncológicas, Madrid 28029, Spain Metabolic dysfunction contributes to the clinical deterio- ration observed in advanced cancer patients and is charac- terized by weight loss, skeletal muscle wasting, and atrophy of the adipose tissue. This systemic syndrome, termed cancer-associated cachexia (CAC), is a major cause of morbidity and mortality. While once attributed solely to decreased food intake, the present description of cancer cachexia is a disorder of multiorgan energy im- balance. Here we review the molecules and pathways re- sponsible for metabolic dysfunction in CAC and the ideas that led to the current understanding. Human cancers develop as a localized focus of uncon- trolled cell growth and subsequently progress to a system- ic disease (Fig. 1). Cancer research primarily focuses on the agents, events, and genetic alterations underlying tu- mor initiation, progression, and metastasis. However, the vastmajority of end-stage cancer patients suffers a sys- temic illness defined as cachexia, a widespread but poorly understood condition (Lok 2015). The “most time hon- ored symptom of cancer” (Editors 1929), cachexia is the prototype image that comes to mind when thinking of cancer. The loss of appetite, energy, and vigor; the languid and unsmiling face; the sallow and anemic aspect; and the skinny and wasted complexion are all too familiar to phy- sicians treating cancer patients. Despite the obvious clin- ical picture, a formal definition of the diagnostic criteria has only recently been reached (Fearon et al. 2011). The current consensus for diagnosis is the unintentional loss of total bodyweight or skeletalmusclemass. Importantly, cancer-associated cachexia (CAC) is a complex metabolic disorder with profound changes in energy balance, which might be already irreversible at the time of obvious body weight loss. While cachexia itself is often rapidly progres- sive, marking the irreversible decline in health and sur- vival, the time when cachexia appears in the clinical history of the cancer patients is, at present, mostly unpre- dictable. The severity of CAC is often unrelated to tumor size or stage, with small tumors commonly leading to severe wasting, as is the case, for example, for pancreatic and lung tumors. In contrast, widely disseminated cancers may cause death without any evidence of CAC. The rea- sons for this paradoxical lack of correlation between tu- mor burden and the degree of cancer cachexia are, at present, elusive. Furthermore, CAC often results in fewer completed cycles of chemotherapy with higher compli- cation rates. Therefore, a better characterization of the metabolic changes in the organism affected by cancer is urgently needed in order to recognize the early events of CAC and improve its prognosis. In this review, we dis- cuss the conceptual advances that shaped the current understanding of the systemic metabolic maladaptation to cancer. Research milestones in cancer cachexia For centuries, the concept that a local malignant growth could be responsible for systemic effects has been under debate (E.F.B. 1909). Rather than a specific disease, the wasting associated with cancer was attributed to nonspe- cific pathological complications of the tumor, such as anorexia, hemorrhage, infection, or ulceration of the neo- plastic tissue (Willis 1948). In contrast, those in favor of systemic alterations produced by the tumor on the host considered cachexia the result of either direct secretion by the tumor of some substances active in distant organs or uptake by the tumor of components from the blood that are essential for the correct functioning of distant organs (Greenstein 1947; Donovan 1954). Evidence supporting one hypothesis or the other was slim, and the contention was disputed on the basis of small clinical case series and anecdotal post-mortem findings (Donovan 1954). During the past decades, a vast body of investigation has reshaped our understanding of CAC (Fig. 2). Experimental work with animal models of cancer rather than [Keywords: cancer-associated cachexia (CAC); metabolic failure; white adipose tissue (WAT) browning; skeletal muscle atrophy] Corresponding authors: ewagner@cnio.es, mp753@cam.ac.uk Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.276733. 115. © 2016 Petruzzelli and Wagner This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full- issue publication date (see http://genesdev.cshlp.org/site/misc/terms. xhtml). After six months, it is available under a Creative Commons Li- cense (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 30:489–501 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/16; www.genesdev.org 489 Cold Spring Harbor Laboratory Press on December 11, 2017 - Published by genesdev.cshlp.orgDownloaded from mailto:ewagner@cnio.es mailto:ewagner@cnio.es mailto:mp753@cam.ac.uk mailto:mp753@cam.ac.uk mailto:mp753@cam.ac.uk http://www.genesdev.org/cgi/doi/10.1101/gad.276733.115 http://www.genesdev.org/cgi/doi/10.1101/gad.276733.115 http://www.genesdev.org/cgi/doi/10.1101/gad.276733.115 http://genesdev.cshlp.org/site/misc/terms.xhtml http://genesdev.cshlp.org/site/misc/terms.xhtml http://genesdev.cshlp.org/site/misc/terms.xhtml http://genesdev.cshlp.org/site/misc/terms.xhtml http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://genesdev.cshlp.org/site/misc/terms.xhtml http://genesdev.cshlp.org/ http://www.cshlpress.com observations in the clinical setting led to the recognition of CAC as a legitimate entity independent of the effects of anorexia or mechanical interference of the tumor with the surrounding tissues. When tumor-bearing rats were force-fed a high-fat diet, weight loss was prevented. However, the development of anemia and the enlarge- ment of the adrenal glands were not affected, thus show- ing the existence of systemic manifestations of cancer independent of nutritional intake (Begg and Dickinson 1951). Following the kinetics of tissue loss in tumor-bear- ingmice, it was noticed that adipose tissuewastingwas an early event, occurring at a time when the tumor was bare- ly palpable (Costa andHolland 1962). Surprisingly, fat loss could also be induced by nonviable tumor preparations, indicating that soluble components of tumor extracts can induce cancer cachexia (Costa and Holland 1962). The causes for the systemic effects associated with cancer weremore sophisticated than just reduced food intake and needed to be sought in the complex relationship between the host and the tumor. Analogies between systemic re- sponses to infectious agents and cancers were noted, in- cluding fever, leukocytosis, and increased serum levels of acute phase response proteins (Rosenthal and Franklin 1975). The first evidence that inflammatory mediators— namely, cytokines—were involved in the process of pro- tein breakdown in isolated skeletalmuscle and a potential role for interleukin-1 (IL-1) in muscle degradation during fever was published in 1983 (Baracos et al. 1983). Particu- lar attention was received by the somewhat paradoxical increase in serum lipid levels despite the obvious loss of body weight in severely sick patients. It was found that such hypertriglyceridemia could be induced experimen- tally in animals by either injection of infective agents or transplantation of tumors (Rouzer and Cerami 1980; Kawakami and Cerami 1981). Hypertriglyceridemia was the result of lipoprotein lipase (LPL) inhibition and could be reproduced by injecting animals with conditioned me- dium from inflammatory cells incubated with endotoxin (Kawakami and Cerami 1981). In 1985, Bruce Beutler in Anthony Cerami’s group (Cerami et al. 1985) provided definitive proof that circulat- ing mediators could cause cachexia, showing that culture medium from endotoxin-activated macrophages caused body weight loss when injected into mice. The molecule in the conditioned medium causing cachexia was purified and termed “cachectin” (Beutler et al. 1985). Subse- quent determination of the complete primary structure of cachectin revealed its identity with tumor necrosis fac- tor-α (TNFα) (Fransen et al. 1985; Pennica et al. 1985). It should be noticed that these early preparations of condi- tionedmedium containedmultiplemacrophage products, and it is therefore erroneous to attribute all of the cachex- iogenic action to the effect of TNFα alone. Furthermore, TNFα causes systemic shock and the release of other cyto- kines, further confounding the attribution of the observed phenotype to a single identifiable factor. However, despite the technical limitations, these early studies contributed to a conceptual evolution in the field of cachexia research, and the wasting syndrome was finally regarded as the re- sult of the host response to the tumor. Clinical evidence confirmed the conclusions of preclinical investigations showing that intravenous hyperalimentation could not al- leviate cachexia in cancer patients (Evans et al. 1985). Re- markably, the focus of research had gradually shifted from the nature of the invasive agent (infection or cancer) to the quality of the response elicited in the organism. The im- mune systemwasthe likely source of allmediators respon- sible for the systemic changes, and a variety of cytokines joined TNFα in the ability to cause systemic alterations (Beutler and Cerami 1986). However, despite the clear role played by cytokines in experimental cachexia, their involvement in human disease was less obvious, and clin- ical translation yielded ambiguous results (Balkwill et al. 1987; Socher et al. 1988). Perhaps as a consequence of the disappointment generated by the lack of clinical benefits from basic findings, the pace of research in the field of can- cer cachexia progressively reduced.An additional explana- tion could be the rapid progress of molecular biology of cancer in themid-1980s. That was the timewhen the first oncogenes were discovered, and the simplistic view of re- ducing cancer to a single base mutation was occupying the entire scene (Weinberg 2014). The war on cancer Figure 1. Multilevel cancer pathophysiology at a glance. Sche- matic representation of the evolution of cancer from a single transformed cell to a systemic disease. At the “cell” level, the transformed cell (purple) is characterized by the presence of ge- nome instability andmutations, sustained proliferative signaling, avoidance of growth suppressors, replicative immortality, resis- tance to cell death, and deregulated cellular energetics. At the “tissue” level, the proliferation of the tumor mass is associated with induction of angiogenesis, activation of invasion, promotion of inflammation, and avoidance of immune destruction—all hall- marks of cancer as described in Hanahan andWeinberg (2011). At the “organism” level, the developing cancer induces changes in distant organs, including metastasis, metabolic failure, and can- cer cachexia, which is not included in the hallmarks of cancer. Cancer cells are shown in purple, “normal” cells are indicated in blue, immune cells are shown in green, and blood vessels are indicated in red. The arrow at the “cell” level points to the con- version from a normal cell to a cancer cell, whereas the arrow pointing to the “organism” depicts a growing tumor (purple). Petruzzelli and Wagner 490 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press on December 11, 2017 - Published by genesdev.cshlp.orgDownloaded from http://genesdev.cshlp.org/ http://www.cshlpress.com seemed to be close to a favorable ending, and the focus of research zoomed back to the tumor itself rather than the response that the tumor ignites in the organism. Cancer cachexia is an energy balance disorder linked to inflammation Weight loss is acardinal signof cachexiaand represents the main independentpredictorofmortality in cancerpatients (Fearon et al. 2012). The mechanisms for weight loss in cancer are multiple, including decreased nutrient intake, systemic metabolic dysfunction, and increased energy ex- penditure. Inflammation represents a common denomina- tor in the pathophysiology of energy imbalance during cachexia. In mice, the peritoneal injection of cancer cells expressing TNFα has been shown to cause weight loss and cachexia. In contrast, mice injected with the same cells without TNFα do not lose body weight (Oliff et al. 1987). Similar results have been obtained with IL-6 in pre- clinicalmodels (Strassmann et al. 1992). Both host- and tu- mor-derived cytokines cooperate in a complex way with the tumor microenvironment to sustain tumor growth andcachexia (Cahlin et al. 2000). In support of a role of can- cer-derived cytokines, it has been shown recently that ex- pression of the cytokine TNF-related weak inducer of apoptosis (TWEAK) by cancer cells causes cachexia, and the effect is similar in wild-type and TWEAK-deficient mice (Johnston et al. 2015). As the cancer persists, it is assumed that ongoing local inflammationmay reach a threshold when cytokines spill into the circulation, thus transforming the cancer disease from a localized tumor to a systemic impairment. Unfor- tunately, such a simplistic view does not stand present ex- perimental validation. The levels of serum cytokines do not correlatewith the appearance of cachexia in cancer pa- tients (Fearon et al. 2012). Furthermore, treatments with antibodies targeting a single cytokine have failed so far to prevent or significantly ameliorate the wasting syn- drome (Penna et al. 2010; Fearon et al. 2013). Very recent data emphasize the multifactorial etiology of CAC, show- ing now that a combination of cytokines and/or additional mediators is responsible for the cachectic phenotype (Schaefer et al. 2016). Despite the absence of a simplistic threshold model linking cytokine levels to cachexia development, a rich body of evidence supports their causal role in the meta- bolic dysfunction observed in CAC. Mechanistically, cytokines were shown to increase the metabolic rate through activation of thermogenesis, inhibit adipocyte and skeletal myocyte differentiation, and reduce food intake (Guttridge et al. 2000; Li et al. 2002; Ruan et al. 2002; Arruda et al. 2010). However, weight loss in cancer patients cannot be attributed solely to decreased food in- take, since dietary supplements fail to reverse cachexia (Bruera and Sweeney 2002). In contrast, a recent study in mice expressing high levels of the proinflammatory cyto- kine IL-18 suggests that high caloric feeding in the context of metabolic dysfunction may exacerbate weight loss and cause fatal cachexia (Murphy et al. 2016). In the context of cancer, metabolic dysfunction is caused by deregulated carbohydrate and lipid metabolism. Altered carbohydrate metabolism in cancer cachexia Carbohydrate intolerance in cancer patients has long been noted (Rohdenburg et al. 1919). While fasting blood sugar concentration between control and cancer groups did not differ significantly, intravenous glucose tolerance tests showed significantly decreased disappearance of glucose in cancer patients (Bishop and Marks 1959). In the first half of the last century, Cori and Cori (1925) compared glucose levels in the venous blood from the tumor-bearing arm and the unaffected arm of a patient with a sarcoma on the forearm. Glucose levels from the tumor-bearing arm were reduced, thus confirming in vivo the increased rate of tumor glycolysis (Cori and Cori 1925). Since tumor tis- sue takes up glucose, the decreased disappearance of glu- cose observed in the tolerance test must be sought in metabolic alterations in the host tissues associated with cancer development. Either increased hepatic glucose pro- duction or a decrease in peripheral utilization could ac- count for the reduced glucose tolerance observed in cancer patients. Despite decreased hepatic glycogen stores, endogenous glucose production is increased in ca- chectic patients due to increased hepatic glucose recy- cling via lactate, a phenomenon termed the Cori cycle 1951 1962 1983 1985 1993 2001 2011 2015 Injection of non-viable tumor preparations causes fat atrophy in mice Cachectin/TNF-α is the first identified mediator of cachexia Myostatin causes skeletal muscle atrophy Anamorelin increases lean body mass in a Phase III clinical trial International consensus on the diagnostic criteria of cachexia Role of the ubiquitin pathway in skeletal muscle atrophy IL1 causes protein breakdown in isolated skeletal muscle Systemic manifestation of cancer despite forced- feeding in rats Figure 2. Timeline of discoveries in cancer cachex- ia. In 1951, the first systemicmanifestation of cancer was described in rats. In 1962, it was observed that injection of tumor preparations in mice was suffi- cient to induce fat atrophy. In 1983 and 1985, the first candidate molecules were identified. Seminal publications in 1993 and 2001 described a role for the ubiquitin pathway and myostatin in skeletal muscle atrophy. It was not until some years ago that an international consensus on the diagnostic criteria of CAC was reached. Promising results have been reported in late 2015 from the first phase III clinical trial targeting CAC with the ghrelin re- ceptor agonist anamorelin (https://www.iaslc.org/ news/results-phase-iii-trials-anamorelin-advanced- non-small-cell-lung-cancer-patients-cachexia). Cancer cachexia and metabolic dysfunction GENES & DEVELOPMENT 491 Cold Spring Harbor Laboratory Press on December 11, 2017 - Published by genesdev.cshlp.orgDownloaded from https://www.iaslc.org/news/results-phase-iii-trials-anamorelin-advanced-non-small-cell-lung-cancer-patients-cachexia https://www.iaslc.org/news/results-phase-iii-trials-anamorelin-advanced-non-small-cell-lung-cancer-patients-cachexia https://www.iaslc.org/news/results-phase-iii-trials-anamorelin-advanced-non-small-cell-lung-cancer-patients-cachexia https://www.iaslc.org/news/results-phase-iii-trials-anamorelin-advanced-non-small-cell-lung-cancer-patients-cachexia https://www.iaslc.org/news/results-phase-iii-trials-anamorelin-advanced-non-small-cell-lung-cancer-patients-cachexia https://www.iaslc.org/news/results-phase-iii-trials-anamorelin-advanced-non-small-cell-lung-cancer-patients-cachexia https://www.iaslc.org/news/results-phase-iii-trials-anamorelin-advanced-non-small-cell-lung-cancer-patients-cachexia http://genesdev.cshlp.org/ http://www.cshlpress.com (Holroyde et al. 1984). Apart from these studies, clinical investigations on glucose metabolism in cachectic pa- tients are noticeably thin. While one study suggests that glucose intolerance may worsen with the development of cachexia (Jasani et al. 1978), other studies found that glucose intolerance did not correlate with body weight loss (Yoshikawa et al. 2001; Agustsson et al. 2011). Very recently, elegant genetic studies in the fruit fly Droso- philahave identified an important role of insulin signaling in inducing a cachexia-like systemic wasting following transplantation ofDrosophila tumors (Figueroa-Clarevega and Bilder 2015; Kwon et al. 2015). Both studies have iden- tified a tumor-secreted factor, ImpL2/IGFBP (an insulin- binding protein and antagonist of insulin/insulin-like growth factor [IGF] signaling), that is responsible for the wasting phenotypes in organs distant from the transplant- ed tumors (Wagner and Petruzzelli 2015). Role of lipids, burning fat, and white adipose tissue (WAT) browning Besides changes in carbohydrate metabolism, the han- dling of lipids between tissues is severely impaired in can- cer patients. The deposition of triglycerides (TGs) in cytoplasmic lipid droplets represents the most efficient form to store lipids inWAT andmany other cell types. Al- ready in 1848, the French physiologist Claude Bernard discovered that TGs, commonly called fat, are digested in the gut before they can be absorbed. The hydrolysis of TGs, designated lipolysis, generates glycerol and fatty ac- ids (FAs). The enzymes mediating intracellular lipolysis include adipose TG lipase (ATGL) and the hormone-sensi- tive lipase (HSL), while LPL is responsible for the hydroly- sis of plasma TGs of lipoproteins in the vascular system (Young and Zechner 2013). FA uptake and TG synthesis decline in WAT in murine cancer models, whereas, in human CAC, it is associated with normal lipid synthesis but elevated lipolysis in WAT. This suggests that lipid ca- tabolism is more relevant than inhibition of lipid synthe- sis for the loss of WAT in CAC (Dahlman et al. 2010). These findingswere corroborated in an elegant study dem- onstrating that WAT lipolysis in cancer patients is in- creased due to elevated enzyme activities of ATGL and HSL (Das et al. 2011). Importantly, genetic deletion of Atgl in mice prevented increased lipolysis and the reduc- tion of WAT and skeletal muscle mass in certain models of CAC. Similar results were also observed, although to a lesser extent, when HSL was inactivated (Das et al. 2011). Lipolysis in CAC is induced bymany serum factors secreted by tumor or host cells, including hormones such as glucocorticoids and catecholamines; cytokines like TNFα, IL-1β, IL-6, prostaglandins; and a zinc–glycopro- tein, ZAG, also called lipid-mobilizing factor (Tisdale 2010). How functional lipolysis impacts the development of cancer cachexia is the focus of ongoing investigations in several laboratories (for review, see Tsoli et al. 2015). While quantitative changes in WAT content during cancer cachexia have long been recognized, only recently a qualitative change in the morphology and function of white adipocytes has been described. During the progres- sion of cancer cachexia in preclinical models, WAT cells gradually convert to brown adipose tissue (BAT)-like cells, also called “beige” cells, in a process termed “browning” (Kir et al. 2014; Petruzzelli et al. 2014). Beige cells are characterized by high mitochondrial content and in- creased expression of uncoupling protein 1 (UCP1), which is responsible for uncoupling the use of mitochondrial electron transport from ATP synthesis to thermogenesis (Nedergaard and Cannon 2014). The phenomenon of browning was initially described as an adaptive response to prolonged exposure to cold environments (Cousin et al. 1992). When exposed to cold temperatures, mice de- ficient in the ability to activate thermogenesis rapidly lose core body temperature and are more susceptible to cold-induced damage (Nguyen et al. 2011). The induc- tion of browning in humans was initially hypothesized on the basis of increased fluorodeoxyglucose (FDG) up- take in WAT depots using positron emission tomography (PET) (Nedergaard et al. 2007) and later confirmed at the histological level (Cypess et al. 2009; vanMarkenLichten- belt et al. 2009; Virtanen et al. 2009). Recent investiga- tions have shown that the role of browning is not limited to cold acclimatization. In preclinical models of diet-induced obesity, browning promotes systemic energy expenditure, which results in body weight loss and improved insulin sensitivity. The protection conferred by browning against high-fat diet-induced obesity sug- gests pharmacological enhancement of browning as a promising therapeutic strategy for metabolic disorders due to excess of nutrients (Feldmann et al. 2009; Yone- shiro et al. 2013). While the effect of browning is identical in both obesity and cancer, the metabolic result is the op- posite. Increased lipid mobilization and energy expendi- ture are favorable in obesity while being deleterious in cancer (Fig. 3). In fact, different from obesity and the met- abolic syndrome, browning in the context of cancer exac- erbates the metabolic dysfunction, enhancing energy dissipation and contributing to the progression of CAC (Kir et al. 2014; Petruzzelli et al. 2014). Browning in can- cer-bearing mice is a systemic event manifested in multi- ple WAT depots. It precedes the onset of skeletal muscle atrophy and determines a hypermetabolic state character- ized by high resting energy expenditure. Notably, brow- ning is not restricted to one experimental model and is not associated with one specific cancer type, since it was documented in complementary model systems, in- cluding genetically engineered mouse models (GEMMs), carcinogen-induced cancers, syngeneic transplants of mu- rine cancer cells, and xenogeneic transplants of human cancer tissue (Petruzzelli et al. 2014). Recently, browning of WAT has been shown to take part in the pathogenesis of hypermetabolism commonly observed in other morbid conditions, like post-burn injury, severe adrenergic stress, and kidney failure (Kir et al. 2015; Patsouris et al. 2015; Sidossis et al. 2015). Treatment of mice with a synthetic thyroid hormone receptor agonist induces adaptive thermogenesis in subcutaneous WAT, thus suggesting a role for WAT browning also in hyperthyroidism (Lin et al. 2015). Petruzzelli and Wagner 492 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press on December 11, 2017 - Published by genesdev.cshlp.orgDownloaded from http://genesdev.cshlp.org/ http://www.cshlpress.com Tumors can directly activate thermogenesis in beige cells through the secretion of parathyroid-related peptide (PTHrP), which has been identified in the supernatants from amurine lung carcinoma cell line and shown to dras- tically induce the expression ofUCP1 (Kir et al. 2014). At the molecular level, transformation of white adipocytes into beige cells requires the function of the transcriptional coregulator PRDM16. Interestingly, fat-specific Prdm16- deficient mice challenged in a model of cancer cachexia showed a significant reduction of browning, thermogenic activity, and WAT atrophy. Importantly, injection of ca- chectic xenotransplant mice with a neutralizing antibody specific for PTHrP was beneficial, reducing the intensity of cancer cachexia and skeletal muscle atrophy. In lung and colorectal cancer patients, higher plasma PTHrP con- centrations are associated with increased energy expendi- ture and enhanced lean tissue wasting, thus confirming the therapeutic potential of inhibiting PTHrP in human cancer (Kir et al. 2014). While treatment of cachectic mice with a PTHrP antibody ameliorated the severity of cachexia, it did not inhibit it completely, thus suggesting that other tumor-derived or host-derived molecules col- laboratewith PTHrP in the induction of browning and sys- temic wasting. Next to direct activation of browning through tumor- derived PTHrP, systemic inflammation and activation of the β-adrenergic pathway represent complementary mechanisms involved in the pathogenesis of browning during CAC (Petruzzelli et al. 2014). Plasma levels of IL- 6 are increased in cachectic mice, and genetic blockade of IL-6 by stable incorporation of a shRNA led to a drastic reduction of the severity of cancer cachexia in a xenogene- ic cancer model. In addition, IL-6 receptor (IL-6-R) knock- out mice implanted with melanoma cells displayed reduced browning when compared with control mice, fur- ther corroborating the role of IL-6 in the activation of the thermogenic program in white adipocytes (Petruzzelli et al. 2014). While the direct induction of UCP1 expres- sion by incubation of adipocytes in the presence of recom- binant IL-6 is modest, indirect mechanisms are likely to enhance IL-6-induced browning, such as alternative acti- vation of macrophages (Mauer et al. 2014). These cells have been shown to sustain adaptive thermogenesis by means of enhanced recruitment of β-adrenergic fibers (Nguyen et al. 2011). Indeed, macrophages infiltrate the WATof cachecticmice and expressmarkers of alternative activation. The link between the immune system and adipose tissue biology is further supported by recent in- vestigations showing WAT browning following micro- biota depletion (Suarez-Zamorano et al. 2015; Yeoh and Vijay-Kumar 2015). Interestingly, colonization of the in- testine by different strains of bacteria has been shown to modulate disease severity and cachexia development in mousemodels (Schieber et al. 2015). A role for therapeutic agents targeting intestinal function inCAC remains large- ly speculative at present (Klein et al. 2013). Whether inhibition of browning may indirectly affect tumor me- tabolism is not known. Systemic alterations of themetab- olism in the host are predicted to affect local metabolic pathways of cancer cells, although more experimental data are needed. In addition to browning, many studies using murine cancer models have demonstrated that lipolysis induces the activation of interscapular BAT during cancer cachex- ia, further contributing to energy uncoupling in mito- chondria with the subsequent worsening of the negative energy balance (Tsoli and Robertson 2012). BAT has a key role in thermogenesis and energy balance and there- fore may well participate in energy expenditure in cancer patients. BAT has been shown to be present in adult hu- mans, and a role for BAT in CAC is possible but is by no means definitively proven (Bauwens et al. 2014). Muscle wasting in cancer cachexia CAC is characterized by muscle atrophy, which severely impairs the patient’s mobility because of fatigue and weakness (Cohen et al. 2015). Early labeling experiments have shown that different mechanisms cause skeletal muscle atrophy in different conditions. Increased myofi- brillar degradation was observed in skeletal muscle White Adipose Tissue (WAT) Beige adipose tissue C el l White Adipocyte Beige Adipocyte Ti ss ue msi nagr O Obesity: beneficial Cancer: detrimental UCP1 Inflammation (IL-6) β-adrenergic stimulation Parathyroid-hormone -related protein (PTHrP) Energy storage Production of heat Lipolysis WAT browning Decreased lipogenesis Mitochondrial content Browning of WAT Figure 3. Mechanisms and consequences of WAT browning in cancer cachexia. At the “cell” level, beige adipocytes are induced in WAT by a combination of signaling pathways, including β-ad- renergic stimulation, inflammation mediated by IL-6, and the presence of parathyroid-related peptide (PTHrP); as a result, UCP1 levels andmitochondrial content are increased. At the “tis- sue” level, CAC is associated with the appearance of islets of beige adipocytes in WAT, surrounded by white adipocytes of re- duced size due to ongoing lipolysis. WAT browning and lipolysis result in decreased energy storage and increased production of heat. In the context of obesity,WATbrowning is beneficial, while in cancer patients, it is detrimental. Cancer cachexia and metabolic dysfunction GENES & DEVELOPMENT 493 Cold Spring Harbor Laboratory Press on December 11, 2017 - Published by genesdev.cshlp.orgDownloaded from http://genesdev.cshlp.org/ http://www.cshlpress.com atrophy caused by denervation,while a combination of de- creased synthesis and increased degradationwas responsi- ble for cortisone-induced muscle atrophy (Goldberg and Goodman 1969). In animal models, glucocorticoids did not cause skeletal muscle atrophy at physiological con- centrations but only at increased concentrations under pathological conditions (Tomas et al. 1979). Therefore, increased adrenal activity and glucocorticoid levels in cancer patients were hypothesized as likely to be respon- sible for skeletal muscle-wasting during cancer cachexia. However, adrenalectomy did not prevent skeletal muscle atrophy in tumor-bearing animals, thus arguing against a role for adrenal hyperfunction in muscle atrophy during experimental CAC (Svaninger et al. 1987). Conversely, microscopic examination of skeletal muscle from cancer cachexia patients did not show evidence of degeneration of muscular or intramuscular nerve bundles, thus exclud- ing also a role for denervation (Marin and Denny-Brown 1962). The factors responsible for skeletal muscle atrophy in CAC remained elusive until the important role of cyto- kines was finally identified. Administration of TNFα or IL-1 in mice was found to cause loss of skeletal musclemass similar to whatwas ob- served in cachectic cancer patients (Fong et al. 1989). However, while treatment of tumor-bearing rats with anti-cytokine immunoglobulins reduced skeletal muscle atrophy, the protection against systemic wasting was only partial (Costelli et al. 1993). Evidence accumulated pointing to the idea that, in cachexia, the synergistic ac- tion of multiple cytokines and other mediators was re- sponsible for skeletal muscle atrophy and likely most of the other components of the wasting syndrome (Argiles and Lopez-Soriano 1999). At themolecular level, the ubiq- uitin-dependent proteasome pathway (UPP) was identi- fied as one important mechanism underlying muscle breakdown in pathologic states, such as prolonged fasting and metabolic acidosis (Wing and Goldberg 1993; Mitch et al. 1994). Similarly, activation of the UPP was observed in preclinical models of cancer cachexia (Temparis et al. 1994; Baracos et al. 1995). At the genetic level, deletion of muscle-specific E3 ligases Atrogin-1/MAFbx or Murf1 (muscle RING finger protein 1) protected skeletal muscle against experimental atrophy (Bodine et al. 2001). In con- trast, muscle-specific activation of NF-kB caused skeletal muscle wasting (Cai et al. 2004). In vitro studies have shown a role for TNFα in the activation of NF-kB, which results in inhibition of myocyte differentiation (Guttridge et al. 2000). In addition, cytokines cause a reduction in myofibrillar protein by decreasing the expression of nucle- ar transcription factor MyoD and through activation of UPP (Acharyya et al. 2004). A large body of evidence im- plicates the FOXO family of transcription factors as key mediators of skeletal muscle atrophy during CAC as well as during fasting and other pathological conditions (Egerman and Glass 2014; Cohen et al. 2015). The catabol- ic effects of FOXO transcription factors are mediated by induction of the atrophy-related ubiquitin ligase Atro- gin-1/MAFbx (Sandri et al. 2004) and Murf1 (Zhao et al. 2007; Cohen et al. 2009). A third E3 ligase, Mul1, has been shown to be involved in the reduction of oxidative capacity in cachectic muscles by controlling mitochon- drial protein degradation (Lokireddy et al. 2012). Compelling evidence shows that the atrophy-related genes, also called atrogenes, are directly responsible for skeletal muscle atrophy due to conditions different from CAC, such as denervation, diabetes, or renal failure. Therefore, this points to a concept that a common tran- scriptional program underlies the loss of skeletal muscle mass independently of the triggering factor (Lecker et al. 2004; Sandri et al. 2006). Skeletal muscle activation of atrogenes in experimental cachexia may also be the result of cross-talk mechanisms between distant organs. As pre- viously noticed, genetic inhibition of lipolysis amelio- rates skeletal muscle atrophy in mouse models of CAC (Das et al. 2011). Lipolysis determines an elevated flux of FAs from adipose tissue, and increased FA uptake in the skeletal muscle leads to ceramide synthesis, reduced mTOR activity, and Atrogin and Murf expression (Cor- coran et al. 2007; De Larichaudy et al. 2012). In this regard, intramyocellar lipid droplets have been described in skel- etal muscle of cancer patients, and its overall content was associated with the extent of body weight loss (Stephens et al. 2011). While there is considerable experimental evidence for the contribution of atrophy-related UPP in preclinical models, its direct role in human disease and human CAC in particular is, at present, controversial. Conflicting evidence comes from studies that have measured the ex- pression levels of the different UPP components in cancer cachexia patients. Arguing against a direct role, individual components of the UPP were actually found to be un- changed or even down-regulated in cancer patients with suppression of both anabolic and catabolic processes, in- dicative of reduced muscle turnover that was restored to normal levels following tumor resection (Stephens et al. 2010; Gallagher et al. 2012). On the contrary, different studies reported an increase in the expression levels of proteasome subunits in skeletal muscle of cancer patients with weight loss (Williams et al. 1999; Khal et al. 2005). Besides overexpression of the ubiquitin gene, direct mea- surement of the proteasome proteolytic activity showed enhancement in skeletal muscles of patients with gastric cancer when compared with noncancer surgical controls and was associated with advanced tumor stage and poor nutritional status (Bossola et al. 2003). These conflicting data comparing animal models and patients with CAC may be due to differences in timing of examination of the skeletal muscle. The analysis in rodents was per- formed during or at the end of rapid skeletal muscle wast- ing, while, in cachectic humans, it was performed in the final stage that follows the period of dramatic wasting. It has been shown that changes in expression levels of atro- genes are maximal during the periods of rapid changes in skeletal muscle mass, while further weight loss is associ- ated with reduced gene expression (Khal et al. 2005). Mul- tiple time points during skeletal muscle atrophy in human cachexia must be measured before conclusions can be drawn. Additional mechanisms of muscle atrophy in cachexia have been suggested, including activation of the JAK/STAT3 pathway (Bonetto et al. 2012; Shum and Petruzzelli and Wagner 494 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press on December 11, 2017 - Published by genesdev.cshlp.orgDownloaded from http://genesdev.cshlp.org/ http://www.cshlpress.com Polly 2012), induction of apoptosis (He et al. 2014), mito- chondrial dysfunction (White et al. 2011), and the direct effect of cancer chemotherapy (Le Bricon et al. 1995). Besides the factors responsible for skeletal muscle atro- phy, studies on factors relevant for muscle hypertrophy have also provided important insights into the mecha- nisms underlying muscle wasting in CAC. Insulin is the main anabolic factor opposing the catabolic effects of glu- cocorticoids, and the absence of insulin in rats contributes to skeletal muscle atrophy (Price et al. 1996). At the mo- lecular level, IGF-1 activates insulin receptor substrate 1, which signals through PI3K–AKT to induce protein syn- thesis by activating mTOR (Rommel et al. 2001). Skeletal muscle hypertrophy is also observed in the presence of in- activating mutations in Myostatin (Schuelke et al. 2004), while forced expression of Myostatin causes muscle atro- phy in adult mice (Zimmers et al. 2002). The muscle hypertrophy observed inmyostatin-deficientmice is abol- ished after inhibition of bone morphogenetic protein (BMP) signaling, which results in up-regulation of the muscle ubiquitin ligase of the SCF complex in atrophy-1 (MUSA1) (Sartori et al. 2013). Myostatin and Activin are members of the transforming growth factor β (TGFβ) fam- ily that were shown to be involved in skeletal muscle atrophy by binding to theMyostatin/Activin type II recep- tor B (ActRIIB) (Benny Klimek et al. 2010). Interestingly, expression of a dominant-negative ActRIIB in transgenic mice results in skeletal muscle hypertrophy (Lee and McPherron 2001). Furthermore, expression of Myostatin is increased upon inflammatory signaling, whereas it inhibits myoblast differentiation and increases Foxo activation and the expression of ubiquitin ligases (Sartori et al. 2009; Trendelenburg et al. 2009). A recently identi- fied PGC1α isoform, Pgc1α4, has been shown to be highly expressed in exercised muscle and was able to prevent skeletal muscle atrophy by repressing Myostatin activity. Notably, mice with skeletal muscle expression of Pgc1α4 were protected from CAC (Ruas et al. 2012). As an additional mechanism for skeletal muscle dysfunction in cancer, TGFβ release from bone metastasis has been demonstrated to lower intracellular calcium signaling and reduce the force of muscle contraction (Waning et al. 2015). From a therapeutic perspective, recent clinical trials have provided proof of principle that it is possible to pro- mote skeletal muscle anabolism in cancer patients. A high-protein diet supplemented with leucine has been shown to increase muscle fractional synthetic rate in a small randomized trial in cancer patients (Deutz et al. 2011). However, it has been reported that leucine supple- mentation increases pancreatic cancer growth in mice, a mechanism mediated by activation of mTOR (Liu et al. 2014). The landmark study by Zhou et al. (2010) has shown that pharmacological blockade of ActRIIB in mouse models of CAC ameliorates skeletal muscle atro- phy and prevents atrophy of cardiac muscle. Importantly, ActRIIB blockade significantly prolonged survival even in the absence of direct effects on tumor growth and cyto- kine secretion. At present, it is not clear whether Myosta- tin inhibition may also ameliorate skeletal muscle atrophy by direct stimulation of stem cell proliferation. Protection against skeletal muscle atrophy and regrowth of skeletal muscle myocytes are observed after ActRIIB blockade, although a causative role of Myostatin inhibi- tion has yet to be proven. Impaired regenerative capacity of myogenic cells has been recently described in CAC, a process mediated by NF-kB-dependent expression of the self-renewing factorPax7 (He et al. 2013). Furthermore, in- hibition of ActRIIB by a humanized monoclonal antibody has been shown to increase skeletal muscle mass and pre- vent glucocorticoid-induced atrophy in mice (Lach-Trifi- lieff et al. 2014). The beneficial effects of inhibiting the Myostatin/Activin pathway is not limited to ameliorating skeletal muscle atrophy but was also shown to improve other pathological conditions in preclinical models, such as insulin resistance and systemic inflammation. The translational potential of Myostatin/Activin antagonism is currently being evaluated in multiple clinical settings (Han et al. 2013; Cohen et al. 2015). Implementation of these findings in clinical practice is anticipated to poten- tially ameliorate the prognosis of cancer patients. The role of the liver in cancer cachexia Next to WAT and skeletal muscle, the liver is of primary importance in the control of systemic metabolism. How- ever, the nature and extent of liver damage during CAC has received little attention. Similarly, the contribution of the liver to the metabolic dysfunction observed in ca- chexia is currently poorly characterized. Enhanced liver inflammation during CAC is suggested by increased infil- tration of macrophages in the livers of pancreatic cancer patients with cachexia when compared with pancreatic cancer patients without cachexia (Martignoni et al. 2009). Activated macrophages in the liver parenchyma may provide a local source of IL-6 production, which stim- ulates the synthesis of hepatic acute-phase protein (Cas- tell et al. 1989). Preclinical investigations have shown that hepatic oxidative phosphorylation is reduced in a rat model of peritoneal carcinosis, concomitant with in- creased energy wasting and production of reactive oxygen species (Dumas et al. 2010). Furthermore, clinical investi- gations have shown that hepatic gluconeogenesis is in- creased in cancer patients (Yoshikawa et al. 1999). Last, hepatic steatosis has been documented in CAC patients (Teli et al. 1995). At the molecular level, hepatic gene ex- pression of the transcription factor TGFβ1-stimulated clone 22 D4 (TSC22D4) is increased in experimental cachexia and correlates with the degree of systemic wast- ing (Jones et al. 2013). Gene expression levels of the nucle- ar receptor cofactor receptor-interacting protein 140 (RIP140) are also increased in CAC and may contribute to liver steatosis by preventing the release of TG stores (Berriel Diaz et al. 2008). Endocrine routes to cancer cachexia Activation of neuroendocrine responses plays amajor role in CAC (Fearon et al. 2012). The role of the hypothalamus Cancer cachexia and metabolic dysfunction GENES & DEVELOPMENT 495 Cold Spring Harbor Laboratory Press on December 11, 2017 - Published by genesdev.cshlp.orgDownloaded from http://genesdev.cshlp.org/ http://www.cshlpress.com in cachexia has been the focus of intense investigations due to the critical function that this gland has in the cen- tral control of food intake and appetite. Cytokine ex- pression in the brain is very low under physiological conditions but is strongly induced in response to peripher- al inflammation and is prominent in the hypothalamus (Grossberg et al. 2009). Feed-forwardmechanisms amplify and maintain the inflammatory response in the brain through local production of cytokines and neurotransmit- ters. Neurons in the arcuate nucleus of the hypothalamus and in the nucleus tractus solitarus of the brainstem com- pose the central melanocortin system (Fan et al. 1997), and inhibition of this neural network has been demon- strated to alleviate the severity of CAC in preclinicalmod- els (Wisse et al. 2001; Cheung et al. 2005). For instance, treatment with the gastrointestinal hormone ghrelin inhibits the central melanocortin system and reduces hy- pothalamic inflammation, resulting inweight gain and in- creased lean body mass in tumor-implanted rats (DeBoer et al. 2007). The central mechanisms responsible for cyto- kine-induced body weight loss include reduction of food intake and increased metabolic rate through activation of thermogenesis (Li et al. 2002; Arruda et al. 2010). Less is known on the role of the hypothalamus in the pathogen- esis of endocrine dysregulation observed in an organism affected by cancer, although a potential contribution of the hypothalamic–pituitary–adrenal axis is suspected. Chronically elevated IL-6 levels in the brain have been shown to activate the hypothalamic–pituitary–adrenal axis and cause adrenal gland hyperplasia (Raber et al. 1997). Furthermore, increased serum levels of hormones from the cortical adrenal gland (cortisol) andmedulla (nor- epinephrine and epinephrine) have been reported in pa- tients with cachexia associated with chronic heart failure, a condition termed cardiac cachexia (Anker and Coats 1999). Conclusions and perspectives Our understanding of CAC has changed dramatically over the past three decades (Fig. 4). It suffices to look at the dif- ferences between reviews published a few decades ago to realize the conceptual leap forward. In 1977, “cancer ag- gression” had a minor metabolic component for cachexia development, which was caused solely by the cancer tis- sue (Costa 1977). The present description envisions CAC as a complex and multifactorial syndrome resulting from the interaction and mutual effects of the tumor and host tissues (Fearon et al. 2012). Experiments in animal models proved instrumental in revealing the mechanisms by which the tumor perturbs host homeostasis—mecha- nisms that reach far beyond reduction of food intake or lo- cal damage at the site of tumor growth. Studies in preclinical GEMMs helped to define the molecular mech- anisms involved in key manifestations of systemic wast- ing. During the past years, many scientists thought that the basic pathological events had been characterized and that the responsible factors had been enumerated. The wealth of knowledge generated on the molecular mecha- nisms underlying CAC pathophysiology has paved the way to novel therapeutic approaches, and new candidate molecules hold promise for clinical use (Table 1). Howev- er, to date, the therapeutic application of basic discoveries has proven elusive (Lok 2015), and current therapeutic management of cachectic patients is palliative, based on appetite improvement and best supportive care (Ma et al. 2014). We now know that prevention and treatment of CAC needs to be multifactorial, as targeting single mediators has repeatedly failed. To increase the chances of success, treatment has to start early in the clinical history of can- cer patients, before obvious evidence of metabolic dys- function. A phase of adapting and adjusting must occur between the tumor and the host in the “unaffected” weight-stable cancer patient. Characterization of these early events at multiple organ levels is essential for un- derstanding the pathophysiology of the host–tumor inter- action, including the neuroendocrine axis (Lainscak et al. 2008). Similarly limited is our knowledge of themetabolic cross-talk between the tumor and the host, which is the starting point for understanding the progression from a local malignant growth to a systemic disease. The characterization of these events requires a new level of “systemic approaches” to design the right experiments for a scientific field that has historically been studying one phenotype or organ and one tumor at the time. Future investigations focusing more on the “whole” and less on the “parts” will go beyond the dichotomy tumor–or- ganism and provide the conceptual framework to devise new therapeutic strategies for treating the organism in ad- dition to just killing the tumor. Such holistic approaches CANCER CACHEXIA Adrenal hypertrophy Liver inflammation WAT atrophy WAT browning Muscle atrophy Heart atrophy Gut barrier disfunction Brain/ Endocrine dysfunction Cytokines and tumor/host-derived factors Figure 4. Conceptual evolution of the understanding of cancer cachexia. The scheme depicts the way we envi- sionmultifactorial cancer cachexia in 2015, involving re- ciprocal compounding interactions between the tumor and the organism, which result in inflammatory and metabolic changes distant from the pathological sites of tumor growth. This way is very different from the uni- directional way that “cancer aggression” was viewed de- cades ago (Costa 1977). Petruzzelli and Wagner 496 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press on December 11, 2017 - Published by genesdev.cshlp.orgDownloaded from http://genesdev.cshlp.org/ http://www.cshlpress.com will likely lead to a better understanding of the metabolic dysfunction in cancer cachexia for the benefit of cancer patients. Acknowledgments We are very grateful to Dr. Doug Hanahan, Dr. Anna Hupalow- ska, Dr. Graham Robertson, Dr. Martina Schweiger, Dr. Marta Shahbazi, Dr. Rudolf Zechner, and our laboratory colleagues Dr. Latifa Bakiri, Dr. Oezge Uluckan, and Dr. Sebastian Hasen- fuss for critical reading and comments on our manuscript. E.F. W. is supported by a grant from the SpanishMinistry of Economy (BFU2012-40230) and a European Research Council Advanced Grant (ERC FCK/2008/37). References Acharyya S, Ladner KJ, Nelsen LL, Damrauer J, Reiser PJ, Swoap S, Guttridge DC. 2004. Cancer cachexia is regulated by selec- tive targeting of skeletal muscle gene products. 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Novel pharmacological agents and potential future treatment strategies for CAC Agent Mechanism of action Physiological effects References Anamorelin Ghrelin receptor agonist Appetite-enhancing and anabolic activity Garcia et al. 2015 Bimagrumab Anti-ActRII monoclonal antibody Prevent skeletal muscle atrophy Lach-Trifilieff et al. 2014 Clazakizumab Anti-IL-6 monoclonal antibody Anti-inflammatory activity Bayliss et al. 2011 Enobosarm Selective androgen receptor modulator Anabolic activity Dobs et al. 2013 IP-1510 IL-1 receptor antagonist Anti-inflammatory activity Paspaliaris et al. 2011 MABp1 Anti-IL-1α monoclonal antibody Anti-inflammatory and anti-neoplastic activity Hong et al. 2014 REGN1033 Myostatin antagonizing antibody Prevents skeletal muscle atrophy Ebner et al. 2014 A selective set of drugs are listed: Anamorelin holds great promise, since, in two phase III clinical trials, a favorable clinical response profile was obtained in patients with cachexia–anorexia and non-small-cell lung (NSCL) cancer. 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