Molecular Mechanisms of Muscle Wasting in Cancer Cachexia

Cachexiais a syndrome associated with cancer that causes -22% of deaths in terminally ill patients. The syndrome is marked by anorexia, anemia, metabolic changes, and muscle wasting. Loss of muscle contributes to reduced quality of life and morbidity. The severity of muscle loss is associated with increased levels of inflammatory cytokines. Tumor necrosis factor-a (TNFa) is considered the classical model of cytokine induced muscle catabolism and is believed to playa major role in the muscle wasting aspect of cachexia. Animal models support this hypothesis and cell culture models show a direct effect of 1NFa on muscle protein breakdown via the ubiquitin (ub)/proteasome pathway. However, little is known about the contribution of other inflammatory cytokines. Interleukins such as Interleukin-6 (IL-6) and Interleukin-1a (IL- 1a) have also been implicated in cancer cachexia. Increased circulating levels of IL-6 is associated with colon, prostate, lung, thyroid, cervical, and pancreatic carcinomas. IL-1a has been implicated in the cachexia of breast cancer, is elevated in pancreatic cancer and is expressed in primary breast, ovarian, and bladder cancer cells and breast cancer cell lines. Although interleukins are implicated in muscle catabolism, only 1 report identifies a direct effect of interleukins on total protein breakdown in an isolated and controlled cell culture system. The goal of this project is to test for a direct effect of interleukins on muscle protein catabolism in cell culture myotubes and to identify cellular and molecular mechanisms by which these effects are mediated. Three Aims will be addressed. Aim 1: To test the effect of interleukins on promotion of muscle specific degradation via the ub/proteasome pathway. Mature myotubes will be derived from the C2C12 myoblast cell line (ATCC, CRL-1772). Myotubes will be exposed to increasing doses of interleukins. Following treatment, total muscle protein and myosin levels will be measured. A decrease in muscle proteins with increasing interleukin dose is expected. Net activation of ub-conjugation will also be measured and is expected to increase with interleukin treatment. Finally, a direct relationship between ub/conjugation and degradation of muscle specific proteins will be tested. Myotubes will be pretreated with 26S proteasome inhibitor (MG-132); control myotubes will be left untreated. Ub-conjugated muscle proteins will be measured and are expected to increase either with interleukin treatment or proteasome inhibitor. Aim 2: To determine interleukin-activated signaling pathways and their contribution to regulation of ub-conjugation. Stimulation of muscle catabolism is believed to involve both Nuclear Factor- KB (NFKB) and mitogen-activated protein kinase (MAPK) signaling. Myotubes will be exposed to interleukins and the activation of these pathways will be determined. In addition, specific inhibitors will be used to test the dependence of ub-conjugation on NFKB and MAPK signaling. Activation of both NFKB and MAPK signaling is expected and inhibitors are expected to block ub-conjugation. Aim 3: To determine ub/proteasome regulatory proteins that are essential for cytokine regulated muscle catabolism. The mRNA for muscle-specific ub regulatory proteins UbcH2 (ub carrier protein), Atrogin and MuRF1 (ub ligases) are upregulated in many models of cancer cachexia. To test for cytokine stimulation of ub regulatory proteins, myotubes will be treated with interleukins and mRNA expression levels will be tested. Specific inhibitors will be used to test the dependence of ub regulatory proteins on NFKB and MAPK signaling. Interleukins are expected to stimulate an increase in mRNA expression that can be blocked by NFKB and MAPK inhibitors. Finally, a direct link between interleukininduced ub-conjugating activity and UbcH2, Atrogin and MuRF1 and will be tested. siRNA technology will be used to knock down expression of these proteins. The gene-specific siRNAs are expected to block interleukin induced ub-conjugating activity.