Molecular Analysis of Xenograft Models of Human Cancer Cachexia

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Abstract. Approximately 50% of all cancer patients develop cachexia, a paraneoplastic syndrome that is characterized by wasting of adipose tissue and skeletal muscle mass. Cytokines, including TNF-·, interleukins-1, -6, and interferon-Á are known mediators of the cachectic process. The latter however represent only one of many imbalanced systems in cancer cachexia. The aim of this study was to further delineate the pathogenesis of cachexia by molecular profiling. Human renal cancer xenografts that do and do not induce cachexia in mice were used as disease models. Cachexia-associated gene expression was studied on Human Genome U95 Affymetrix arrays and revealed several new genes such as TNF-· ligand superfamily protein, interferon-Á treatment inducible protein, and DKFZ5641I1922. The expression of the IL-8 gene was also elevated in cachexia inducing xenografts (CIX). At the protein level, TNF-· was found expressed only in CIX, whereas IL-1 and IL-6 were not cachexia specific. Levels of parathyroid hormone-related protein were elevated in CIX and accompanied by hypercalcemia. COX-2 and prostaglandin E2 were also found to be over expressed. By using the COX-2 inhibitors rofecoxib and nimesulide, we were able to delay tumor-mediated wasting in vivo. Overall, our results suggest that cachexia is a multigenetic disease that will require complex combinations of drugs for an effective therapeutic intervention. Cancer is frequently associated with anorexia, progressive weight loss and accelerated malnutrition resulting in the depletion of whole body lipid and protein stores. This body wasting condition, named cachexia, is accompanied by poor quality of life, poor response to chemotherapy and reduced survival time irrespective of tumor mass or the presence of metastases. The pathogenesis of cancer-associated cachexia is not fully understood, however, multiple mediators including neuroendocrine hormones, tumorspecific factors and pro-inflammatory cytokines may be involved. Although experimental evidence strongly supports a role of the pro-inflammatory cytokines TNF-·, interleukin-1 (IL-1) and interleukin-6 (IL-6) in the etiology of cachexia, a causative function for cytokines in cachexia development remains controversial. While a clear association between induction of cachexia and cancer exists for TNF-· (also termed cachexin), IL-1 appears not to be necessary for inducing cachexia, and IL-6 is not active on its own, but only in concert with other cachectic factors. Prostaglandins, particularly prostaglandin E2 (PGE2), are key down-stream effecter molecules of cytokine activity. Inhibitors of the PGE2-signalling pathway exist and have been used to prove the involvement of cytokines in cachexia development. Thus, by blocking PGE2 activity with specific cycloxygenase (COX) inhibitors, responses were noted in tumor-bearing mice. Anorexia, cachexia and even tumor progression were markedly attenuated in murine tumor models of cachexia with meloxicam and indometacin. As a result, a clinical trial in patients with advanced cancer was initiated using indometacin. Anti-TNF-· antibodies were also studied, but with only limited success and were not further pursued. More recently treatment with IGF- 1, owing to its prominent role in muscle metabolism, has been proposed as a promising approach for the prevention of cancer-related muscle atrophy. However, targeted treatments remain experimental at best and current therapies aiming to alleviating cancer cachexia are mostly based on nutritional approaches. Hence, a better knowledge of the molecular pathways governing cachexia is required in order to develop more effective treatments. Interferon-Á treatment inducible protein is a proinflammatory peptide which is induced by interferon-Á. The fact that IFN-Á is considered a cachectic factor and together with TNF-· may be responsible for the lowering of muscle protein syntheses, this peptide could be another cachexiaassociated cytokine. Little is known about the function of DKFZ564I1922. However, it has been detected in the cartilage of patients with arthritis. Because arthritis is a severe inflammatory disease, DKFZ564I1922 might be a cachexia-mediating cytokine. The results described in this study, demonstrate that the development of experimental cachexia by human renal tumor xenograft models is more complex than previously described in reports on the role of pro-inflammatory cytokines such as TNF-·, IL-1, or IL-6. Our experiments demonstrate that the cachectic syndrome represents a multifactorial, severe inflammatory event in which a number of different mediators play a role. Immunological parameters appear useful in assessing the conditions of the disease as well as for a therapeutic intervention. An effective treatment of cancer cachexia will however require complex combinations of drugs that target pro-inflammatory cytokine networks. While our renal cell cancer xenograft models were very homogenous in their gene and protein expression profiles, it is possible that other tumor histologies show a different molecular fingerprint of cachexia mediating factors. Our studies should thus be extended to and validated with clinical tissue specimens. If our findings are confirmed, cachexia-associated gene profiles could be used for early detection of the syndrome in individual patients, and COX-2 inhibitors together with therapeutic antibodies against pro-inflammatory cytokines could be administered to intercept cancer cachexia development in its early stages.