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Cancer cachexia is a chronic wasting syndrome characterised by loss of weight, composed principally of muscle and fat. Patients with advanced cachexia demonstrate loss of appetite, early satiety, severe weight loss, weakness, anaemia and fluid retention. Affected individuals are also likely to report/experience decreased quality of life, decreased levels of physical performance, increased levels of fatigue, increased risks of treatment failure (be it chemotherapy, radiotherapy or surgery), increased risks of treatment side effects, and an increased mortality rate. Cachexia is therefore an extremely important, yet often underappreciated cause of cancer patient morbidity and mortality which requires urgent attention. Weight loss is significantly associated with cancer morbidity and mortality. It has been observed that half of all cancer patients experience weight loss and one-third lose more than 5% of their original body weight. Skeletal muscle loss appears to be the most significant event in cachexia and is associated with a poor outcome. However it is not known why some patients with the same tumour lose weight and muscle mass whilst others do not. The main aim of this thesis was to determine if the genetic makeup of individual patients might contribute to their propensity to lose weight or skeletal muscle. Previous studies had suggested an association between weight loss and SNPs on genes concerned with innate immunity and particularly the cell adhesion molecule Pselectin, however the strength of any gene association study depends on the precision with which it is possible to characterise the phenotype in question. A second aim of this thesis was to explore refining the clinical phenotyping of patients to discriminate those with evidence of muscle fibre atrophy versus those without. Phenotype The conventional phenotype for cachexia is weight loss (WL) but it is unknown the extent to which loss of body mass reflects loss of muscle or fat mass. Recent progress in cross sectional imaging analysis means that it is now possible to gain a direct measure of muscle mass from routine diagnostic CT scanning. However, in the absence of a longitudinal series of scans it is not possible to estimate whether low muscularity (LM) is longstanding or not. By combining a measure of active weight loss with low muscularity it was hoped that such a composite measure would reflect actual muscle loss/fibre atrophy. Compared with non-cachectic cancer patients, patients with LM or LM+ > 2%WL, mean muscle fibre diameter was reduced by about 25% (p = 0.02 and p = 0.001 respectively). No significant difference in muscle fibre diameter was observed if patients had WL alone. Regardless of classification, there was no difference in fibre number or proportion of fibre type across all myosin heavy chain isoforms. Mean muscle protein content was reduced and the ratio of RNA/DNA decreased in patients with either > 5%WL or LM+ > 2%WL. These findings support the use of composite measures (WL and LM) to try and identify those patients with evidence of active muscle fibre atrophy. This novel clinical phenotyping provides an accurate method to enable the conduct of candidate gene studies in the investigation of the genetics of cancer cachexia where the primary focus is on muscle wasting rather than overall weight loss. Genotype In an ideal world it would be possible to explore the entire genome and look for associations with the different phenotypes of cachexia. However, to do so would require considerable resource in terms of the cost of genome wide analysis and the cost of phenotyping large enough cohorts of patients (3000-10000). To address these issues I therefore adopted a candidate gene approach. A total of 154 genes associated with cancer cachexia were identified and explored for associated polymorphisms. Of these 154 genes, 119 had a combined total of 281 polymorphisms with functional and/or clinical significance in terms of cachexia associated with them. Of these, 80 polymorphisms (in 51 genes) were replicated in more than one study with 24 polymorphisms found to influence two or more hallmarks of cachexia (i.e. inflammation, loss of fat mass and/or lean mass and reduced survival). Such election of candidate genes and polymorphisms is a key element of multigene study design. The systematic review provides a contemporary basis to select genes and/or polymorphisms for further association studies in cancer cachexia, and to develop their potential as susceptibility biomarkers of cachexia. Phenotype – genotype associations A total of 1276 patients were recruited, phenotyped and genotyped. There were 545 new patients and 731 patients from a previous study. In our new cohort and in keeping with the previous literature, patients who carried the C allele of the rs6136 SNP in the SELP gene, were at a reduced risk of developing cachexia defined by WL. This association applied to all degrees of weight loss ( > 5%, > 10% or > 15%), and not just at the > 10% level as described previously in the literature. When examining newly identified SNPs in a stage 1 analysis for the weight loss phenotype that included 1276 cancer patients, twelve new candidate SNPs were significant. Six of these SNPs are associated with muscle metabolism in five genes (IGF1, CPN1, FOXO1, FOXO3, and ACVR2B), three are associated with adipose tissue metabolism in two genes (LEPR and TOMM40 (APOE on the reverse strand)), two with corticosteroid signalling in one gene (IFT172 (GCKR on the reverse strand)) and one with the immune response in one gene (TLR4). Two polymorphisms (rs1935949 and rs4946935) in the gene encoding for FOXO3 were consistently associated with WL of increasing severity ( > 5% and > 10%). On the basis that WL is a continuum in the cachectic process, the observation that both SELP and FOXO3 associate with the higher degrees of WL suggests that these genetic signatures may be of particular significance. The role of P-selectin in the genesis of cachexia remains to be determined. When examining all SNPs in a stage 1 analysis for the LM phenotype, 5 SNPs were associated significantly with the cachexia phenotype: (i) rs4291 in the angiotensin converting enzyme (ACE) gene in chromosome 17; this gene has been associated with muscle function and metabolism; (ii) rs10636 in chromosome 16 in the metallothionein 2a gene; this gene has been shown to be involved in zinc dyshomeostasis which may contribute to cancer cachexia; (iii) rs1190584 in chromosome 14 in the WDR20 gene; this gene encodes a WD repeat-containing protein that functions to preserve and regulate the activity of the USP12-UAF1 deubiquitinating enzyme complex; (iv) rs3856806 in the peroxisome proliferator-activated receptor gamma (PPARG) gene in chromosome 3 which has been demonstrated to be involved in fatty acid and glucose metabolism; and (v) rs3745012 in chromosome 18 in the lipin 2 (LPIN2) gene; this gene represents a candidate gene for human lipodystrophy, characterised by loss of body fat, fatty liver, hypertriglyceridemia, and insulin resistance. When examining all SNPs in a stage 1 analysis for the LM + > 2%WL phenotype 4 SNPs were associated significantly with the cachexia phenotype. rs12409877 in the leptin receptor (LEPR) located on chromosome 3, LEPR binds leptin and is involved in adipose tissue regulation. rs2268757 located in the activin receptor type-2B (ACVR2B) gene on chromosome 3, ACVR2B is a high affinity activin type 2 receptor which mediates signalling by a subset of TGF-β family ligands including myostatin, activin, GDF11 and others. SNPs in the tumour necrosis factor (TNF) (rs1799964) and ACE (rs4291) genes were also significantly associated with the phenotype. Whether genes demonstrating significant associations with the cachexia phenotypes had altered transcript expression in muscle from cancer patients with or without those phenotypes was also investigated.

This work examines skeletal muscle catabolism in cancer and its attenuation by Eicosapentaenoic Acid (EPA). In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma - MAC16, demonstrated an elevation in the gastrocnemius muscle in the activity and expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. This was accompanied by an accelerated loss of muscle tissue correlating with an increase in overall weight loss, all of which were attenuated by prior daily dosing with EPA. Recently a proteolysis inducing factor (PIF) has been isolated from the MAC16 tumour, and from the serum and urine of cachectic cancer patients. Previous studies have shown that PIF induces protein degradation in vitro, and that this is possibly mediated through 15-hydroxyeicosatetraenoic acid (15-HETE), a metabolite of the n-6 polyunsaturated fatty acid- arachidonate. Employing the murine myoblast cell line C2C12, it was shown that both PIF and 15-HETE increased protein degradation and expression of proteasome subunits, processes which were again attenuated by prior incubation in EPA. Similarly, in NMRI mice which had been fasted for 24hours, EPA and the lipoxygenase inhibitor CV-6504 (but not structurally related fatty acids) inhibited skeletal muscle proteolysis and expression of various proteasome subunits, showing that firstly, EPA may be anti-cachexic partly through its ability to influence 15-HETE production; and secondly that the effect is specific for EPA as other fatty acids had no effect. Previous studies have suggested the involvement of the signal transduction family NFKB in response to PIF in the liver. It has been demonstrated here that both PIF and 15-HETE increased nuclear translocation of NFKB in the skeletal muscle of tumour bearing mice and that EPA inhibited this process by its ability to prevent the degradation of the NFKB inhibitor protein IKB. When an NFKB inhibitor was added to C2C12 myotubes, prior to the addition of PIF, proteasome activity and protein degradation was inhibited, showing that NFKB is responsible for the increased proteasome activity and muscle catabolism induced by PIF. Taken together this work suggests that 15-hydroxyeicosatetraenoic acid is the intracellular mediator for PIF induced protein degradation in skeletal muscle and that elevated muscle catabolism is accomplished through an increased functioning of the ubiquitin-proteasome pathway, a process possibly mediated through an NFKB dependent mechanism. The anticachectic (and possibly the anti-tumourigenic) effects of EPA appear to be achieved in part by its ability to inhibit the degradation of IKB and possibly by its ability to interfere with 15-HETE production.

Thesis (Ph. D.)--University of Sydney, 2009. / Title from title screen (viewed Aug. 11, 2009) Includes tables and questionnaires. Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Surgery, Faculty of Medicine. Degree awarded 2009 ; thesis submitted 2008. Includes bibliographical references. Also available in print form.

Cancer cachexia is a complex syndrome that can affect up to 80% of cancer patients. Among the symptoms involved in cancer cachexia progression, the establishment of a systemic inflammation and the imbalance in glucose metabolism homeostasis take an important part in this profile. The aim of the present study was to further evaluate the role of cancer-induced inflammation in the impairment of pancreatic beta cell function in solid Ehrlich carcinoma-bearing mice. For that, we have focused the study in the pro-inflammatory mechanisms involved on β-cell death. We have observed that tumor-bearing animals developed an aggressive pancreatic inflammatory status 14 days after tumor cells inoculation. The increase of pro-inflammatory cytokines followed by an up-regulation of important transcription factors such as NF-κB and STAT-1 and its related genes, reveled a similar outline for β-cell death found in type 1 diabetes. Furthermore, expression of pro-apoptotic Bcl-2 family members followed by caspases activation was increased in pancreatic islets of tumor-bearing animals and the expression of anti-apoptotic members was decreased. We have also observed an increase in β-cell death and ER stress components, as well as a decrease in insulin content cells together with an increase in alpha cells content. Overall, our results provide strong evidences that pancreatic β-cells in tumor-bearing animals are widely affected by tumor presence and systemic inflammation establishment. Interestingly, it was shown a similarity with mechanisms of β-cell death found in type 1 diabetes. Although the exactly mechanisms behind the changes found in carbohydrate metabolism in cancer cachexia is still unclear, our data can help to clarify, at least in part, this profile and would serve as a basis for development of new strategies to prevent cachexia progression and to improve the quality of life of cancer patients. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

CCAAT/Enhancer Binding Proteins are a family of six bZIP transcription factors. C/EBPβ, the second member cloned, has been implicated in adipogenesis and osteogenesis, but the role of C/EBPβ in myogenesis remained undetermined. In adults, muscle-resident stem cells, called satellite cells (SCs), have the greatest propensity to regenerate the skeletal muscle. We found that C/EBPβ is expressed in SCs, and its expression progressively declines upon differentiation. Forcing the expression of C/EBPβ in myoblasts enhanced the expression of the SC marker Pax7, and repressed MyoD and the myogenic genes expression, resulting in the inhibition of myogenesis. Using a SC-specific conditional knockout (cKO) mouse model, we found that cKO myoblasts have decreased expression of Pax7, and we identified Pax7 as a direct target of C/EBPβ action. In vivo, excision of C/EBPβ resulted in muscle hypertrophy at the juvenile age, and adult cKO animals had enhanced muscle regeneration following BaCl2 muscle injury. Moreover, the number of Pax7+ cells in cKO animals decreased following BaCl2 injury. Upon performing a second injury into cKO animals, we demonstrate a decreased muscle fiber size and an exacerbation of the percentage number of SCs. While cKO animals repaired well a BaCl2 injury, regeneration failed in cKO animals following cardiotoxin (CTX) injury. We demonstrate that IL-1β expression is enhanced in muscle after CTX injury when compared to BaCl2, and we found that IL-1β can stimulate the expression of C/EBPβ in myoblasts. Ectopic C/EBPβ expression can protect myoblasts from apoptosis when triggered with thapsigargin, whereas cKO myoblasts are more sensitive to apoptosis. Using cancer cachexia as a model of chronic inflammation, we found that the expression of C/EBPβ is stimulated in the SCs of cachectic animals, and this correlated with a decrease in regenerative capacity. The severity of muscle wasting was not improved in cKO animals, but rather cKO SCs were lost to apoptosis. Together, this study establishes a protective role for C/EBPβ in muscle SCs in conditions of inflammation.

Cancer cachexia is a debilitating, paraneoplastic syndrome commonly associated with late stage malignancy. It is estimated that ~25% of cancer-related deaths are due directly to complications arising from cachexia (Barton, 2001). Cachexia manifests as severe body wasting, primarily due to the loss of skeletal muscle mass. This study tested the hypothesis that muscle atrophy associated with cancer cachexia could be attenuated by using a unilateral, functional overload (FO) model applied concurrently with tumor development. To accomplish this, Morris hepatoma MH-7777 cells were implanted in adult female, Buffalo rats (n = 12) and allowed to incubate for 6 weeks. FO surgeries (n = 12) were performed five days prior to MH-7777 cell implantation. Over the course of six weeks, healthy, age, sex and strain-matched, vehicle-injected rats (n = 12) gained ~5% of body weight compared to tumor-bearing rats that lost ~6% of body weight when adjusted for tumor mass. Tumor-bearing animals experienced significant atrophy to gastrocnemius, tibialis anterior, extensor digitorum longus, plantaris and diaphragm muscles. FO successfully reversed plantaris muscle atrophy in cachectic, tumor-bearing rats (n=5). FO plantaris masses were ~24% larger than contralateral controls. However, this hypertrophic response was not as great as FO plantaris muscles from healthy, sham-operated controls (~44% larger than contralateral controls, n=5). FO plantaris muscles from tumor-bearing rats had ~1.5 fold increase in myonuclei/fiber ratios compared those of sham-operated, tumor-bearing controls (n = 6). Therefore, cancer cachexia did not prevent myonuclear accretion necessary for skeletal muscle hypertrophy. Little data exists on adaptations to myosin heavy chain (MHC) isoforms in cachectic skeletal muscle. Plantaris muscles from tumor-bearing rats displayed decreased percentages of MHC type I compared to plantaris muscles from vehicle-injected controls (7% vs. 3%, respectively). However, FO plantaris muscles from tumor-bearing rats had an increased percentage of MHC type I and decreased percentage of MHC type IIb compared to sham-operated tumor-bearing rats, adaptations commonly seen in trained muscles. Therefore, cancer cachexia did not prevent the capability of skeletal muscle to respond normally to hypertrophic stimuli. This study also attempted to characterize a mechanism responsible for the hypertrophic response, increased myonuclei/fiber ratio and transition toward a slower MHC profile in FO plantaris muscles from tumor-bearing rats. Recently, the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, has been suggested as a critical factor regulating skeletal muscle growth and fiber-type dependent gene expression (Chin, 1998; Wu, 2000; Olson, 2000; Otis, 2001). The protein content of the catalytic subunit (CaNa) and the regulatory subunit (CaNb) of calcineurin were unchanged in plantaris muscles from tumor-bearing animals compared to healthy controls. Furthermore, total and specific (normalized to CaNa protein content) calcineurin phosphatase activity were not altered in any group. Therefore, calcineurin activity did not appear to be associated with the regulation of the morphological and physiological response of hypertrophying plantaris muscles in cachectic, tumor-bearing rats. Overall, this study indicated that atrophied plantaris muscles from tumor-bearing animals have a reduced capacity to hypertrophy potentially due to a decreased myonuclei/fiber ratio. Furthermore, it is unlikely that changes to mass and MHC isoform expression are associated with calcineurin phosphatase activity. / Ph. D.

A caquexia associada ao câncer é uma síndrome multifatorial e multiorgão de etiologia desconhecida caracterizada por profunda perda de massa corporal. Assim, o presente estudo investigou o processo inflamatório sistêmico e no tecido adiposo subcutâneo e testou a hipótese de atenuação da inflamação pelo TF crônico em pacientes com caquexia associada ao câncer e controles. Pacientes foram distribuídos em seis grupos: Controles (Control SED e TR), portadores de tumor gastrintestinal sem caquexia (WSC SED e TR) e portadores de tumor gastrintestinal com caquexia (CC SED e TR). Observamos cessação da perda de massa corporal e ganho cardiorrespiratório (incremento de 209%) entres os pacientes caquéticos treinados e redução de colesterol total no CC TR, de LDL no CC SED e de HDL no CC SED e CC TR. O TF foi capaz de aumentar a concentração de HDL ao longo da intervenção (de 34,83 ± 4,74 mg/dL para 57,0 ± 3,42 mg/dL). Não encontramos diferenças entre as células imunitárias infiltradas no TASC dos pacientes avaliados. Esse é o primeiro estudo, de nosso conhecimento, que demonstra que o exercício físico realizado de forma crônica é capaz de reduzir a inflamação em pacientes com câncer caquéticos, atenuando os sintomas da síndrome. / Cancer cachexia is a multifactorial and multiorgan systemic syndrome with an unknown aetiology and characterized by a profound weight loss. Therefore, this study aimed to investigate the inflammation process in a systemic context and in the subcutaneous adipose tissue (SAT) and to evaluated the possible anti-inflammatory effects of the aerobic exercise in subjects with cancer cachexia. Patients were separated in six groups: Control (sedentary SED and trained TR), weight-stable patients with gastrointestinal cancer (WSC SED and TR) and patients with gastrointestinal cancer and cachexia (CC SED and TR). Cessation of weight loss and cardiorespiratory gain wre observed in the CCTR group and a reduction of total cholesterol in the CCTR group decreased in LDL in the CC SED group and HDL in the CC SED and CC TR groups. Physical exercise was able to increase serum HDL during the intervention. No differences were found in the immune cells infiltrating in the patients sAT (macrophages and MDSC). Inflammatory mediators changed by cachexia were modulated by ET. To our knowledge, this is the first study to demonstrate a reduction of inflammation and cachexia symptoms in cancer patients.

A ausência de terapias eficazes para a caquexia permanece como um problema central para o tratamento do câncer no mundo. Em contrapartida, o treinamento de força (i.e. também conhecido como treinamento resistido) tem sido amplamente utilizado como uma estratégia não farmacológica anticatabólica, prevenindo a perda da massa e da função da musculatura esquelética. Entretanto, o papel terapêutico do treinamento de força na caquexia do câncer permanece apenas especulativo. Portanto, nesse estudo avaliamos se o treinamento de força poderia atenuar a perda da massa e da função da musculatura esquelética em um severo modelo de caquexia do câncer em ratos. Para isso, ratos machos da linhagem Wistar foram randomizados em quatro grupos experimentais: 1) ratos sedentários injetados com solução salina na medula óssea (Controle); 2) ratos injetados com solução salina na medula óssea e submetidos ao treinamento de força (Controle + T); 3) ratos sedentários injetados com células do tumor Walker 256 na medula óssea (Tumor); e 4) ratos injetados com células do tumor Walker 256 na medula óssea e submetidos ao treinamento de força (Tumor + T). Foram avaliados a massa e a área de secção transversa da musculatura esquelética, marcadores de disfunção metabólica e do turnover proteico, a função da musculatura esquelética in vivo e ex vivo, o consumo alimentar, o crescimento tumoral e a sobrevida dos grupos experimentais com tumor. O grupo Tumor apresentou atrofia muscular após quinze dias da injeção das células tumorais como pode ser observado pela redução na massa dos músculos Plantaris (- 20,5%) e EDL (-20%). A atrofia no músculo EDL foi confirmada por análises histológicas, demonstrando uma redução de 43,8% na área de secção transversa. Embora o treinamento de força tenha aumentado o conteúdo proteico da lactato desidrogenase e revertido totalmente o conteúdo da forma fosforilada de 4EBP-1 (i.e. repressor da transcrição de mRNA), ele não atuou na morfologia da musculatura esquelética nos animais com tumor. Além disso, o treinamento de força não atenuou a perda de função da musculatura esquelética, a anorexia, o crescimento tumoral ou a taxa de mortalidade. Contudo, a força muscular, avaliada pelo teste de 1RM, apresentou uma correlação negativa com a sobrevida dos animais (p = 0,02), sugerindo que a perda de força prediz a mortalidade nesse modelo experimental de caquexia do câncer. Em suma, a injeção de células do tumor Walker 256 na medula óssea induz caquexia do câncer em ratos. O treinamento de força não foi eficaz em atenuar a perda de massa e função da musculatura esquelética nesse modelo. Entretanto, a força muscular prediz a sobrevida dos animais, sugerindo que novos estudos são necessários para elucidar o possível efeito terapêutico do treinamento de força para atenuar a caquexia do câncer e a progressão tumoral / The lack of therapies for cachexia is a key problem in cancer treatment. In contrast, resistance exercise training (RET) has been adopted as nonpharmacological anti-catabolic strategy, preventing muscle wasting and muscle dysfunction. However, the role of RET to counteract cancer cachexia is still speculative. Presently, we test whether RET would counteract skeletal muscle wasting in a severe cancer cachexia rat model. Methods: Male Wistar rats were randomly assigned into four experimental groups; 1) untrained control rats injected with saline solution in the bone marrow (control), 2) rats injected with saline solution in the bone marrow and submitted to RET (control + RET), 3) untrained rats injected with Walker 256 tumor cells in the bone marrow (tumor) and 4) rats injected with Walker 256 tumor cells in the bone marrow and submitted to RET (tumor + RET). Skeletal muscle mass and fiber cross sectional area, markers of metabolic and protein turnover impairment, in vivo and ex vivo skeletal muscle function, food intake, tumor growth and mortality rate were assessed. Results: Tumor group displayed skeletal muscle atrophy fifteen days post tumor cells injection as assessed by Plantaris (-20.5%) and EDL (-20.0%) muscle mass. EDL atrophy was confirmed by histological analysis, showing 43.8% decline in the fiber cross sectional area. Even though RET increased the lactate dehydrogenase protein content and fully restored phosphorylated form of 4EBP-1 (i.e. a repressor of mRNA translation) to the control levels in skeletal muscle, it failed to rescue muscle morphology in tumorbearing rats. Indeed, RET has not mitigated loss of muscle function, anorexia, tumor growth or mortality rate. However, loss of strength capacity (assessed by 1-RM test performance) demonstrated a negative correlation with rats´ survival (p = 0.02), suggesting that loss of strength capacity predicts cancer mortality. Conclusions: Bone marrow injection of Walker 256 tumor cells in rats induces cancer cachexia. RET is ineffective to mitigate cancer-induced skeletal muscle wasting in this rat model. However, strength capacity predicts cancer survival, suggesting that new studies are needed to elucidate the putative therapeutic role of different exercise training regimens in counteracting cancer cachexia and tumor progression