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Chapter II: Mechanisms of cancer cachexia

Kenneth Fearon

Cancer cachexia is a multifactorial syndrome with anorexia and a progressive loss of adipose tissue and skeletal muscle mass (Fearon and Strasser et al., 2011), and this is reflected in the complex nature of the underlying mechanisms. The fundamental mechanism driving the negative energy and nitrogen balance in patients with cancer cachexia is a variable combination of reduced food intake and abnormal metabolism. Despite recent progress in elucidating the molecular mechanisms affecting the brain and peripheral tissues, there is still considerable headway to make in understanding all aspects of this distressing disease state. Many of the advances have been in model systems of cancer cachexia, so there is still a need to translate these findings into human cachexia. This lack of understanding, compounded by the complexity and heterogeneity of the disease, has hindered the search for effective treatments, although there has also been advancement in this area, as discussed in Chapter V. In the present chapter we describe the emerging understanding of the mechanisms of cancer cachexia.

Cancer cachexia etiology

A combination of the following factors are thought to be key to the etiology of cancer cachexia (Figure 3) (Donohoe et al., 2011):

Tumor factors. Tumor cells produce pro-inflammatory factors and procachectic factors.

The host response. This includes activation of the systemic inflammatory response, metabolic, immune and neuroendocrine changes.

In addition, a number of other factors can influence the disease (Fearon et al., 2012), including:

Cancer therapy. Surgery, radiotherapy, and chemotherapy can have a detrimental effect on a patient’s nutritional intake as a consequence of the development of systemic inflammation and side effects, including nausea, vomiting, mucositis, taste change, lethargy and depression (Bozzetti, 2010; Donohoe et al., 2011; Skipworth et al., 2006). In addition, patients may be fasted for prolonged periods prior to certain investigations or major surgery.

Tumor characteristics. The tumor site may directly effect food intake, for example a tumor in the upper gastrointestinal tract may lead to obstruction and some cancers can cause discomfort including abdominal fullness and constipation. In addition, the tumor type is important, as cancer cachexia is seen more frequently in patients with advanced lung, pancreatic, prostate, gastric and colon cancers (Del Ferraro et al., 2012).

Figure 3. The primary mechanisms of cancer cachexia and clinical consequences. The tumor, the host response and host–tumor interactions induce pro-inflammatory and pro-cachectic factors, an acute phase response and neuroendocrine dysregulation, which cause metabolic dysregulation leading to cachexia. Reproduced from Donohoe et al., 2011.

Patient-specific factors, including:

Genetic factors. Even among cancer patients with the same tumor type and burden there is variability in who will develop cancer cachexia, which may relate to host genotype (Johns et al., 2014; Tan et al., 2011). Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation among people. Although the vast majority of SNPs have no functional effect, some occur within a gene or in a regulatory region near a gene, and may play a role in disease by affecting the gene’s function. A number of candidate gene SNPs with functional significance for cachexia (inflammation, loss of fat mass and/or lean mass and reduced survival) have been identified (Johns et al., 2014; Tan et al., 2011), but their impact needs to be explored further.

Pre-treatment body mass index. Obesity is associated with a higher risk of developing cancer, however there appears to be an “obesity paradox” with cancer cachexia, whereby obesity becomes protective against cachexia, perhaps due to increased adipose and lean tissue reserves (Fearon et al., 2012). Having said that, some overweight patients hide gross muscle wasting under a layer of adipose tissue. For example, approximately 40% of overweight or obese patients with advanced pancreatic cancer have significant skeletal muscle wasting, a condition that is an independent adverse prognostic indicator (Tan et al., 2009).

Hypogonadism. Weight loss and loss of muscle mass are greater in male than female cancer patients (Baracos et al., 2010), which may be associated with a high prevalence of hypogonadism in males (Skipworth et al., 2011). Up to 50% of men with metastatic cancer prior to chemotherapy have low concentrations of testosterone (Chlebowski and Heber, 1982), reductions of which are associated with reduced bone mass and muscle strength in both men and women (Lobo, 2001; Zitzmann and Nieschlag, 2000). Low concentrations of testosterone are suggested to be contributors to cachexia-related wasting of skeletal muscle (Burney et al., 2012; Evans et al., 2008). However, there are conflicting reports regarding a correlation between body composition (including muscle mass) and the concentration of anabolic hormones (Chlebowski and Heber, 1982; Garcia et al., 2006; Hein et al., 1995; Lenk et al., 2010).

Inflammation is a driving force in cancer cachexia, playing a pivotal role in key aspects of the cancer cachexia phenotype including: systemic inflammation; control of muscle and adipose tissue metabolism and function; control of central energy balance; and modulation of appetite (Seelaender et al., 2012). A complex interplay of the following pro-inflammatory and procachectic factors has been implicated in the etiology of cancer cachexia.

Pro-inflammatory cytokines. Pro-inflammatory cytokines are now widely acknowledged as important players in cachexia, acting both in a paracrine and an endocrine manner. A number of cytokines including interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interferon gamma (IFN-γ), TWEAK, and leukemia inhibitory factor (LIF) are implicated in the etiology of cancer cachexia (Argiles et al., 2009; Fearon et al., 2012; Johns et al., 2013; Onesti and Guttridge, 2014; Seelaender et al., 2012; Seto et al., 2015).

Oxidative stress and reactive oxygen species (ROS). Various studies have shown the presence of oxidized proteins and suggest that antioxidant administration ameliorates some of the key factors of cancer cachexia such as anorexia, reductions in lean body mass, systematic inflammation and fatigue, suggesting an important role of ROS in cancer cachexia (Mantovani et al., 2012). Among other roles, ROS are considered to be important players for muscle protein catabolism in cachexia, via their stimulation of the ubiquitin–proteasome pathway (UPP) (Russell et al., 2007).

Eicosanoids. Eicosanoids are biologically active lipids derived from the arachidonic and other omega-6 unsaturated fatty acids. The metabolism of arachidonic acid by cyclooxygenase (COX), lipoxygenase (LOX) and P-450 epoxygenase pathways generates a substantial number of omega-6 eicosanoids, which include prostanoids, leukotrienes, hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids (Kowalczewska et al., 2010). Both COX and LOX pathways have proinflammatory mechanisms induced by cytokines, and there is evidence that activation of these particular pathways results in chronic inflammation and carcinogenesis, and may contribute to the cancer cachexia (Wang and Dubois, 2010).

Adipokines. These are a specific type of cytokine derived from adipose tissue. They regulate many physiological processes including metabolism, food intake, and inflammation. Adipose tissue expression and plasma concentrations of certain adipokines (including leptin, visfatin, resistin and adiponectin) are altered in cancer cachexia, possibly contributing to cachexia-related inflammation (Kemik et al., 2012; Lira et al., 2011; Nakajima et al., 2010; Seelaender et al., 2012; Smiechowska et al., 2010), a mechanism that has been proposed in obesity (Ouchi et al., 2011).

Pro-cachectic factors. These include proteolysis-inducing factor (Todorov et al., 1996) and lipid-mobilizing factor, which serve to break down proteins and fat, respectively (Donohoe et al., 2011; Onesti and Guttridge, 2014).

Hormones. Steroid hormones, including testosterone and glucocorticoids (Burney et al., 2012; Evans et al., 2008; Morley et al., 2006; Seelaender and Batista, 2014) and appetite modulating hormones including leptin and ghrelin have been implicated in the cancer cachexia syndrome (Argiles et al., 2014; Argiles and Stemmler, 2013; Inui, 2001; Molfino et al., 2014a).

Pathophysiology of cancer cachexia

Cancer cachexia is a syndrome involving many organs and tissues including adipose tissue, brain, liver, gut and heart; however, skeletal muscle tissue, which represents over 40% of total body weight, is perhaps the most significant cachexia target (Argiles et al., 2014; Fearon et al., 2012). Key processes involved in cancer cachexia include systemic inflammation, alterations in the metabolism of skeletal muscle protein and adipose tissue, and reduced appetite, as described in more detail in the following sections.

Systemic inflammation

Systemic inflammation is a hallmark of cancer cachexia, which is indicated by the onset of the acute phase response (APR) (Aoyagi et al., 2015). The APR is a complex early-defense system activated by inflammation and other factors, including infection, stress, trauma and neoplasia (Cray et al., 2009). It involves an increase in the concentration of serum proteins with the aim of restoring homeostasis and promoting healing (Cray et al., 2009). These so-called acute response proteins include C-reactive protein (CRP) (Cray et al., 2009), and in fact, serum CRP is the most widely accepted index of systemic inflammation (Fearon and Strasser et al., 2011). The APR (measured using plasma CRP levels) has been associated with inflammation and weight loss in cachexia (Scott et al., 1996; Staal-van den Brekel et al., 1995), together with reduced quality of life and shortened survival in cachexia patients (Barber et al., 1999; Blay et al., 1992; Deans and Wigmore, 2005; Falconer et al., 1995; O’Gorman et al., 1998).

The pro-inflammatory cytokine IL-6 together with the transcription activator STAT3 have been implicated in mediating the APR in cancer cachexia (Bonetto et al., 2011). The exact mechanisms linking the APR and cachexia are unknown; however, one theory relates to the mismatch in amino acid composition between skeletal muscle and acute phase proteins: up to 2.6 g of muscle protein or 12 g of muscle tissue may be mobilized to synthesize 1 g of APR proteins (Reeds et al., 1994). Therefore, during low food intake this may increase the need for muscle mobilization (Reeds et al., 1994), accelerating muscle wasting in cachectic cancer patients (Fearon et al., 2012).

Skeletal muscle metabolism

Patients with cancer cachexia experience a combination of muscle wasting, reduced strength/physical activity and fatigue, which together contribute to a marked reduction in quality of life. Previously, it was assumed that there was a clear linear relationship between muscle mass and strength in cancer cachexia and that the functional sequelae for patients were mainly the result of reduced muscle mass. It appears, however, that muscle mechanical quality may be altered (Stephens et al., 2012). Moreover, the results of recent phase III intervention trials with muscle anabolic agents also point to a disconnect between muscle mass and function (Fearon et al., 2015). The present discussion focuses on the determinants of reduced myofibrillar mass as this is the area where there is the most evidence to discuss.

Muscle mass generally remains constant in adults in the absence of stimuli (e.g., exercise), reflected by a balance of protein synthesis and degradation (Tisdale, 2009). In cachexia, a decrease in protein synthesis (anabolism), an increase in protein degradation (catabolism), or a combination of both leads to muscle atrophy (Guttridge, 2006). A recent study in patients with cachexia secondary to upper GI malignancy has emphasized that due to the long-time course of wasting in humans with cancer, the differences between synthesis and degradation only need to be small and that synthesis seems to be relatively well preserved (MacDonald et al., 2015). Muscle mass may also be influenced by decreased regenerative capacity and there is some evidence that regeneration may be abnormal in cancer cachexia (Talbert and Guttridge, 2016).

The proteolytic and autophagic pathways operating in skeletal muscle that may be altered during cachexia (Lenk et al., 2010) include:

Ubiquitin–proteasome pathway (UPP). The UPP is the principal mechanism for protein catabolism in the mammalian cytosol and nucleus. In this highly-regulated protein degradation pathway, the target protein is tagged by the covalent attachment of multiple ubiquitin molecules. The attachment of ubiquitin to target proteins requires a series of ATP-dependent enzymatic steps by E1 (ubiquitin activating), E2 (ubiquitin conjugating) and E3 (ubiquitin ligating) enzymes. This process marks the protein for subsequent degradation by the 26S proteasome, composed of the catalytic 20S core and the 19S regulator. This is thought to be the main protein degradation pathway operating during the acute skeletal muscle loss in murine models of cancer cachexia (Lenk et al., 2010). The evidence implicating this pathway in the more chronic cancer-associated muscle loss observed in humans is more controversial, but includes the up-regulation of various components of the UPP (Bossola et al., 2001; Bossola et al., 2003; DeJong et al., 2005; Khal et al., 2005; Sun et al., 2012; Williams et al., 1999; Yuan et al., 2015).

Caspase-dependent apoptosis. Apoptosis is a regulated biochemical process that commits a cell to death. A common feature of cells undergoing apoptosis is the activation of caspases, which are a family of aspartic acid-directed proteases. Caspase-mediated proteolysis of specific proteins results in an irreversible commitment of cells to undergo apoptosis characterized by cytoplasmic shrinkage, membrane blebbing, nuclear condensation, and DNA fragmentation. There are multiple lines of evidence that apoptosis contributes to skeletal muscle wasting during cachexia. For example, tumor-bearing cachectic mice demonstrate an increase in the activity of several caspases (Belizario et al., 2001); and the expression of BARD1 (a protein marker of apoptosis) correlates with increased DNA fragmentation during cachexia in tumor-bearing rats (Irminger-Finger et al., 2006). In addition, apoptosis is increased in skeletal muscle of cachectic gastrointestinal cancer patients, as demonstrated by an increase in muscle DNA fragmentation and in poly (adenosinediphosphate ribose) polymerase (PARP) cleavage (Busquets et al., 2007).

Calcium-dependent calpain system. The calpain protein degradation pathway is composed of two enzymes i.e. calpains, which are calciumdependent, non-lysosomal cysteine proteases, and calpastatin, an endogenous inhibitor, which regulates their activity (Teixeira Vde et al., 2012). Calpains do not degrade muscle proteins directly, but cleave the proteins to be degraded further by another cell proteolysis system. The calpain inhibitor calpastatin is decreased in skeletal muscle and heart of tumor-bearing rats (Costelli et al., 2001), and calpain mRNA is increased in another tumor rat model (Temparis et al., 1994). In addition, muscle protein loss in the AH-130-bearing rats is associated with increased activity of both the ATP-ubiquitin- and the calpain-dependent proteolytic pathways (Costelli et al., 2002). However, overall the evidence for a role of calpains in cancer cachexia is still limited.

Lysosomal. Lysosomes contain a large variety of hydrolytic enzymes that degrade proteins taken in by endocytosis. Lysosomes are responsible for the degradation of long-lived proteins and for the enhanced protein degradation observed under starvation conditions. Cathepsins L, B, D and H are major lysosomal proteases. While some studies have found increases in skeletal muscle cathepsin protein, mRNA and enzyme activity in cancer cachexia (Baracos et al., 1995; Fujita et al., 1996; Lecker et al., 2004; Lorite et al., 1998; Tardif et al., 2013; Tsujinaka et al., 1996), another study found that lysosomal inhibitors were not effective at attenuating the increased rate of skeletal muscle protein degradation, and there were no changes in cathepsin B mRNA and activity (Temparis et al., 1994).

Autophagic-lysosomal. In this pathway portions of the cytoplasm and cell organelles are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the proteins are digested (Lum et al., 2005). Autophagy functions as a stress response that is up-regulated by starvation, oxidative stress, or other harmful conditions. A number of studies have implicated the activation of the autophagic-lysosomal pathway in cancer cachexia (Johns et al., 2013), with a recent study showing that autophagic lysosomal degradation is induced in three different models of cancer associated muscle atrophy and in glucocorticoid-treated animals (Penna et al., 2013).

Figure 4. Alterations in skeletal muscle metabolism during cancer cachexia. (a) Alterations in skeletal muscle metabolism leading to loss of skeletal muscle mass include increased apoptosis, proteolysis and branched-chain AA (BCAA) oxidation, and decreased protein synthesis, amino acid (AA) transport and regeneration; (b) During these processes a number of signaling pathways are activated (shown in purple) by pro-inflammatory cytokines, myostatin and tumorderived factors such as proteolysis-inducing factor (PIF). Protein degradation is increased via nuclear factor-κB (NF-κB) by activating the forkhead (FOXO) family transcription factors, resulting in increased transcription of genes encoding ubiquitin ligases (muscle atrophy F-box protein (MAFBX) and muscle RING finger-containing protein 1 (MURF1)) that are involved in the proteolysis of myofibrillar proteins. PIF and cytokines also activate the p38 and Janus kinase (JAK) MAPK pathways leading to increased caspase activity and apoptosis. Insulin-like growth factor 1 (IGF1), which normally stimulates protein synthesis via AKT and mTOR, is decreased (shown in blue) thereby suppressing protein synthesis. In addition, myostatin can also decrease protein synthesis via SMAD2 and AKT and can activate both protein degradation (via FOXOs) and apoptosis (via the MAPK cascade). Overexpression of peroxisome-proliferatoractivated receptor-γ-co-activator 1α (PGC1α) results in increased expression of genes linked to mitochondrial uncoupling and energy expenditure (such as uncoupling proteins (UCPs). ACTRIIA/B, activin receptor type IIA/B; IGF1R, IGF1 receptor; IκB, inhibitor of NF-κB; IKK, IκB kinase; P, phosphorylation; PIFR, PIF receptor. Reproduced with permission from Argiles et al., 2014.

These pathways all involve a complex interplay of signaling molecules that have been implicated in cancer cachexia including myostatin, activin A, JAK2, STAT, ERK, FOXO, NF-kappaB (NF-κB), AKT and IGF (Figure 4) (Argiles et al., 2014; Fearon et al., 2012). Below we describe in more detail the role of the NF-κB, IGF-1 and myostatin/activin pathways.

NF-kappaB

The NF-κB family is composed of five related transcription factors: p50, p52, RelA (p65), c-Rel and RelB. These proteins function as dimeric transcription factors that regulate the expression of genes influencing a broad range of biological processes including innate and adaptive immunity, inflammation, stress responses, B-cell development, and lymphoid organogenesis. NF-κB has long been considered a prototypical pro-inflammatory signaling pathway, largely based on the activation of NF-κB by pro-inflammatory cytokines such as IL-1, TNF-α, and the role of NF-κB in the expression of other pro-inflammatory genes including cytokines, chemokines, and adhesion molecules. There is extensive evidence for the involvement of NF-κB in multiple mechanisms of the cachectic process (Li et al., 2008; Srivastava and Dhaulakhandi, 2013). With regard to protein degradation, NF-κB stimulates transcription of one of the muscle-specific E3 ubiquitin ligases MuRF-1 (Li et al., 2008; Srivastava and Dhaulakhandi, 2013). 

IGF-1

The IGF-1 pathway increases skeletal muscle mass via stimulation of protein synthesis and inhibition of protein degradation. By contrast, myostatin signaling (see below) negatively regulates skeletal muscle mass by reducing protein synthesis and increasing protein degradation. The effects of IGF-1 on skeletal muscle are mainly mediated by the PI3K/AKT pathway leading to the downstream activation of protein synthesis (Rommel et al., 2001) and inhibition of FOXO-1, which regulates the expression of the musclespecific E3 ubiquitin ligases MuRF-1 and MAFbx (also known as atrogin-1), thereby reducing proteasomal protein degradation (Lenk et al., 2010; Sandri et al., 2004). As such, FOXO-1 silencing has been shown to prevent protein degradation in a murine model of cancer cachexia (Liu et al., 2007). Muscle expression of IGF-1 mRNA decreases progressively in the rat AH-130 hepatoma ascites model of cancer cachexia, whereas IGF-1 receptor and insulin receptor mRNA levels increase compared with controls (Costelli et al., 2006). In addition, circulating levels of IGF-1 and insulin are reduced in this model of cancer cachexia. However, administration of exogenous IGF-1 to tumor-bearing rats did not prevent cachexia (Costelli et al., 2006). Other studies in experimental animal models of cancer cachexia have found an increase in AKT phosphorylation during cachexia (Penna et al., 2010; White et al., 2011; White et al., 2013), despite a reduction in IGF-1 expression (White et al., 2011; White et al., 2013), perhaps reflecting the complexity in the regulation of this pathway. Interestingly, two recent studies report that interference of insulin signaling in the fruit fly Drosophila melanogaster induces systemic organ wasting reminiscent of human cachexia (FigueroaClarevega and Bilder, 2015; Kwon et al., 2015; Wagner and Petruzzelli, 2015). In a drosophila model of cancer cachexia Figueroa-Clarevega and Bilder identified the insulin growth factor binding protein (IGFBP) homolog ImpL2, an antagonist of insulin signaling, as a secreted factor mediating wasting (Figueroa-Clarevega and Bilder, 2015). They reported that ImpL2 is sufficient to drive tissue loss, and insulin activity is reduced in peripheral tissues of tumor-bearing hosts. In addition, knocking down ImpL2 specifically in the tumor ameliorates the wasting phenotype. In a different drosophila model of cancer cachexia, Kwon et al. also identified ImpL2 as a mediator of systemic organ wasting (Kwon et al., 2015). It will therefore be of interest to characterize the role of IGFBP in other model systems of cancer cachexia, and of course in the human disease. Also of interest is that recent studies have identified several microRNAs (miRNAs; non-coding RNAs that play key roles in the regulation of gene expression) that can modulate the muscle IGF1–AKT pathway, as well as the myostatin pathway (Hitachi and Tsuchida, 2013). The evidence for a role of miRNAs in cancer cachexia is still in its infancy (Acunzo and Croce, 2015; Hitachi and Tsuchida, 2013), but they are certainly plausible candidates.

Myostatin and activin

Myostatin, which belongs to the TGF-β superfamily, signals through the activin type II receptors (ActRIIA and ActRIIB). It is mainly secreted from skeletal muscle fibers and acts as a negative regulator of skeletal muscle mass (Sharma et al., 2015). Myostatin gene inactivation induces skeletal muscle hypertrophy (Grobet et al., 2003; McPherron et al., 1997), whereas forced overexpression of myostatin induces skeletal muscle atrophy (Amirouche et al., 2009; Durieux et al., 2007; Zimmers et al., 2002). Elevated levels of myostatin mRNA and protein expression as well as enhanced myostatin signaling have been shown to correlate with cancer-associated cachexia in several experimental models of cancer cachexia (Bonetto et al., 2009; Chacon-Cabrera et al., 2014; Costelli et al., 2008; Liu et al., 2008; Sharma et al., 2015; Zhou et al., 2010). In a study by Gallot et al., myostatin gene inactivation prevented the severe loss of skeletal muscle mass induced in mice engrafted with Lewis lung carcinoma (LLC) cells or in ApcMin/+ mice, an established model of colorectal cancer and cachexia (Gallot et al., 2014). Myostatin loss also reduced the growth of LLC tumors and the number and the size of intestinal polyps in ApcMin/+ mice, strongly increasing survival in both models. Gene expression analysis in the LLC model showed this phenotype was associated with reduced expression of genes involved in tumor metabolism, activin signaling, and apoptosis.

Other studies in mouse models of cancer cachexia have also demonstrated that administration of a soluble form of ActRIIB preserves skeletal muscle mass (Benny Klimek et al., 2010; Zhou et al., 2010), restores muscle strength (Busquets et al., 2012; Zhou et al., 2010) and increases lifespan (Zhou et al., 2010). Targeting ActRIIB ligands can also effect activins, including activin A (Zhou et al., 2010), another member of the TGF-β family induced by inflammatory cytokines implicated in the pathogenesis of muscle wasting in cancer cachexia (Chen et al., 2014; Han et al., 2013; Togashi et al., 2015). Several studies have analyzed myostatin levels in patients with cancer, with conflicting results; these inconsistencies may be the result of differences related to the type of cancer, the stage of cachexia, and/or different laboratory techniques (Sharma et al., 2015). The role of myostatin, and of activin A, in cancer cachexia in humans remains to be clarified. Recent clinical intervention studies have not been informative.

Mechanistically, myostatin and activin A bind to the ActRIIB receptor complex on the muscle cell membrane, which then recruits and activates an Alk family kinase, resulting in the activation of a Smad2 and Smad3 transcription factor complex (Han et al., 2013; Sharma et al., 2015). This, in turn, stimulates FOXO-dependent transcription and enhanced muscle protein breakdown via the UPP and autophagy (Han et al., 2013; Sharma et al., 2015). In addition, Smad activation inhibits muscle protein synthesis by suppressing AKT signaling (Han et al., 2013; Sharma et al., 2015).

Adipose tissue metabolism

Lipid metabolism

In cancer cachexia, skeletal muscle loss is accompanied by extensive loss of white adipose tissue (WAT). The processes (Figure 5) that may be altered in cancer cachexia are (Argiles et al., 2014; Argiles et al., 2005):

Lipolytic activity. Activation of hormone-sensitive lipase in adipose tissue results in increased lipolytic activity and the subsequent release of both glycerol and fatty acids. In addition, there is a decreased anti-lipolytic effect of insulin on adipocytes, together with an increased responsiveness to catecholamines and atrial natriuretic peptide, which stimulate lipolysis.

Lipoprotein lipase (LPL). LPL is an enzyme responsible for the cleavage of both endogenous and exogenous triacylglycerols, which are present in lipoproteins, into glycerol and fatty acids. The activity of the enzyme allows for the entry of fatty acids into WAT, and a decrease in LPL activity therefore results in reduced lipid uptake into the WAT.

De novo lipogenesis. This is an enzymatic pathway for converting dietary carbohydrate into fatty acids that are then esterified to storage triacylglycerols. This process is reduced in tumor-bearing states in mice and humans, resulting in decreased esterification and consequently, decreased triacylglycerol deposition.

Increased production of lipolytic factors by adipose tissue and the tumor, such as IL-6, TNF-α or zinc-α2 glycoprotein contributes to aberrant lipid metabolism and increased lipolysis in cancer cachexia (Agustsson et al., 2007; Tisdale, 2005). Adipose tissue wasting appears to precede skeletal muscle protein degradation both in cancer patients and experimental models of cachexia (Batista et al., 2013; Bing, 2011; Murphy et al., 2010). In this respect, WAT actively expresses and secretes a plethora of pro-inflammatory factors including the adipokines leptin, adiponectin, TNF-α, IL-6, IL-10, plasminogen activator inhibitor-1 and visfatin (Batista et al., 2012a; Batista et al., 2012b; Batista et al., 2013). WAT may therefore play an important role in the characteristic systemic inflammation in cancer cachexia (Batista et al., 2013; Tsoli et al., 2014).

Figure 5. Alterations in adipose tissue metabolism during cancer cachexia. Adipose tissue wasting results from increased lipolysis, decreased lipogenesis from glucose and impaired entry of fatty acids owing to decreased activity of lipoprotein lipase (LPL). In addition, a process known as white adipose tissue (WAT) browning can occur, whereby WAT cells acquire some of the molecular properties of brown adipose tissue (BAT) cells, notably the induction of UCP1 (uncoupling protein 1) expression and the presence of multilocular lipid droplets and multiple mitochondria, resulting in heat production and energetic inefficiency. Inflammatory mediators, such as interleukin-6 (IL-6), and tumor-derived compounds, such as parathyroid-hormone related protein (PTHRP) are implicated in the browning process. Circled “+” symbols indicate pathways that are activated during cachexia.

LMF, lipid-mobilizing factor; Pi, inorganic phosphate. Reproduced with permission from Argiles et al., 2014.

Increased energy expenditure: White adipose tissue (WAT) browning

There are two types of adipose tissue: WAT, which stores excess energy in the form of triacylglycerols, and brown adipose tissue (BAT), which is a key site of heat production (thermogenesis) (Figure 5) (Lo and Sun, 2013). Brown adipocytes in BAT have an extremely high number of mitochondria that contain uncoupling protein-1 (UCP1). Upon activation by long-chain fatty acids, UCP1 increases the conductance of the inner mitochondrial membrane to make BAT mitochondria generate heat rather than ATP, which is distributed to the rest of the body through the circulation. Activation of thermogenesis in the interscapular BAT has been observed in a syngeneic mouse tumor transplant model and suggested to contribute to the hypermetabolic state of cachexia (Tsoli et al., 2012).

When subjected to certain stimuli, specific depots of WAT can also take on a BAT phenotype, a process known as WAT browning (Figure 5) (Argiles et al., 2014; Harms and Seale, 2013). These adipocytes, termed beige or ‘brite’ (brown in white), have many similarities to adipocytes in BAT, including their multilocular lipid droplet morphology, high mitochondrial content and the expression of a core set of brown fat-specific genes including Ucp1. Evidence from Petruzzelli et al. suggests that WAT browning takes place in the initial stages of cachexia and contributes to increased energy expenditure and lipid mobilization (Petruzzelli et al., 2014). Increased formation of beige cells was observed in several different mouse models of cancer cachexia, indicating that it is a consistent feature of cachexia. An increase in lipid mobilization and energy expenditure occurred despite decreased locomotor activity, suggesting that WAT browning, together with enhanced thermogenesis in interscapular BAT, contributes to excessive substrate oxidation and wasting in cancer cachexia. Consistent with a role of inflammation in cancer cachexia, IL-6 signaling and β-adrenergic activation were implicated in the WAT browning phenotype observed in mouse models of cachexia. Importantly, increased UCP1 staining in adipose tissue from cachectic cancer patients was observed, indicating the potential (although still tenuous) importance of WAT browning in human cancer cachexia.

Further evidence for a role of WAT browning in cachexia came from a study by Kir et al. (Kir et al., 2014), who identified tumor-induced factors that increased WAT browning in mouse models of LLC. Parathyroid-hormone related protein (PTHrP) potently induced gene expression of markers of WAT browning and energy metabolism in adipocytes, and injection with a PTHrPneutralizing antibody in cachexia-associated cancer mice prevented cachexia and skeletal muscle atrophy. PTHrP signals through the PTH receptor, a G protein-coupled receptor (GPCR) that activates the cyclic-AMP-dependent protein kinase A (PKA) pathway, as do β-adrenergic receptors. PKA inhibitors indicated that activation of PKA downstream of GPCRs was required for the induction of thermogenic gene expression in primary white and brown adipocytes in culture. Cancer patients with detectable levels of PTHrP had reduced lean body mass and increased energy expenditure compared with patients without detectable levels of PTHrP, confirming an association between elevated levels of PTHrP and tissue wasting and hypermetabolism.

Anorexia

The two driving forces behind weight loss in cancer cachexia are reduced food intake and abnormal metabolism. Both factors contribute to a variable extent in the vast majority of patients (Fearon et al., 2013) and it is a mistake to underestimate the importance of anorexia in the genesis of the cachectic state. Cancer-associated anorexia is common in the advanced stages of many cancers, often compounded by therapy-induced side effects, depressed motor activity, possible obstruction in the gastrointestinal tract by a tumor, and psychological factors. In addition to the effect of cytokines on skeletal muscle, cytokines also act in the hypothalamus to inhibit orexigenic (appetite stimulating) and stimulate anorexigenic (appetite-suppressing) regulatory pathways. In addition, the hormones ghrelin and leptin are also crucial in regulating appetite and may play a role in cancer-associated anorexia and cachexia (Argiles et al., 2014; Argiles and Stemmler, 2013; Inui, 2001; Molfino et al., 2014a).

Ghrelin and leptin

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. It is primarily secreted by the stomach into the bloodstream under fasting conditions, where it signals hunger to the brain. In the arcuate nucleus of the hypothalamus, ghrelin stimulates the production of orexigenic mediators such as neuropeptide Y, γ-aminoisobutyrate and agouti-related gene peptide, and blocks anorexigenic mediators such as pro-opiomelanocortin, α-melanocyte-stimulating hormone and cocaine- and amphetamine-regulated transcript (Shioda et al., 2008). In contrast, leptin, a hormone released by adipocytes, stimulates the anorexigenic pathway and inhibits the orexigenic pathway in the arcuate nucleus of the hypothalamus (Figure 6) (Inui, 2001). Fasting decreases leptin and increases ghrelin production, leading to the activation of the orexigenic pathway (Figure 6). Paradoxically, ghrelin levels are elevated in cancer cachectic patients with neuroendocrine, gastric and lung tumors (Karapanagiotou et al., 2009; Kerem et al., 2008; Takahashi et al., 2009; Wang et al., 2007); this phenomenon has been suggested to be due to “ghrelin resistance”, although these elevations may be a compensatory response reflecting the negative energy balance state (Argiles et al., 2014). In addition to its role in appetite, ghrelin may exert other effects related to cancer cachexia: ghrelin has anti-inflammatory actions, reducing the production of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and increasing circulating IL-10, an anti-inflammatory cytokine (Gonzalez-Rey et al., 2006; Wu et al., 2007). In addition, it also has direct effects on muscle cells by inhibiting the increased protein degradation that is promoted by catabolic cytokines (Reano et al., 2014; Sheriff et al., 2012), and can inhibit apoptosis that is induced by doxorubicin (an antitumoral agent) in skeletal muscle cells (Yu et al., 2014). Ghrelin also has effects on fat storage; it activates white adipocytes (Tschop et al., 2000) while inactivating brown adipocytes, thereby contributing to decreased energy expenditure (Mano-Otagiri et al., 2010). Furthermore, ghrelin also stimulates gastric emptying and acid secretion (Peeters, 2003), which may be important since abnormal intestinal function is often observed in cancer patients.

Figure 6. The anorexigenic and orexigenic actions of leptin and ghrelin. Leptin, a hormone released by adipocytes, operates as a feedback loop to maintain constant stores of fat; it is produced as a function of the amount of fat, and reduces food intake by acting on two hypothalamic pathways. It stimulates an anorexigenic pathway and inhibits an orexigenic pathway, both of which originate in the arcuate nucleus (ARC) of the hypothalamus and project to the paraventricular nucleus (PVN) and the lateral hypothalamic area (LHA). In contrast, ghrelin, which is released by the stomach, acts as an orexigenic molecule. In addition, ghrelin has anabolic effects, stimulating both energy gain and the secretion of growth hormone (GH) by acting directly on the anterior pituitary. Fasting decreases leptin and increases ghrelin production, leading to the activation of the orexigenic pathway, a response that might be important for adaptation to fasting. Ghrelin is also produced by hypothalamic cells, but it is unknown whether this source has a similar action to ghrelin produced by the stomach. GHRH, growth-hormone-releasing hormone. Reproduced with permission from Inui, 2001.

Conclusion

Cancer cachexia is a complex syndrome where differences between murine models and clinical findings have often obscured understanding of the fundamental mediators and mechanisms relevant to the treatment of human disease. We are slowly getting closer to comprehending the dominant mechanisms involved in human cancer cachexia, in the hope of making advances in the assessment (Chapter IV), and management and treatment (Chapter V) of patients.