Veränderungen des Skelettmuskels bei Tumorkachexie Morphologische, metabolische und Genexpressions-Analyse des humanen Musculus rectus abdominis

The complex syndrome of cancer-associated cachexia, an unintentional loss of muscle (and fat) mass, presents an oncological challenge: It reduces the patients’ quality of life and mobility and may lead to death e.g. via respiratory muscle insufficiency. 20% of all carcinoma patients are estimated to die of cancer cachexia, yet neither is the underlying mechanism fully understood nor is the condition therapeutically controllable. Studies on human tissue samples, in particular on skeletal muscle, are scarce and have yielded ambiguous results in contrast to those in cell cultures and animal models. Pancreatic carcinoma is the clinically most impressive example of a malignancy with the most severe and rapid cachexia development. Therefore the aim of this thesis was to evaluate the cancer cachexia-associated changes in skeletal muscle biopsies from patients with pancreatic cancer at the time of their first surgery. The analyses comprised muscle fiber morphometry, the concentration of metabolically important amino acids and the expression of pro-inflammatory, -angiogenetic, -atrophic and -apoptotic signals on RNA as well as protein levels. The study included intraoperative skeletal muscle biopsies from the rectus abdominis muscle of 36 patients with histologically confirmed ductal pancreatic carcinoma. The group of patients with cachexia (N=16) was compared with that without cachexia (N=22, control group). Cancer cachexia was defined as unintentional weight loss of at least 10% over the past 6 months. Transverse cryosections of rectus abdominis muscle were stained for myofibrillar ATPase to measure size, density and composition of muscle fibers, for CD31 to measure capillary contacts and for haemalum to measure centralized nuclei. In addition, the intracellular concentration of 26 amino acids was analyzed by HPLC, differential expression of 29 genes was determined via qRT-PCR, and pools of tissue samples were screened for 102 proteins using a protein array. Contrary to expectations, no significant decrease in muscle fiber size or increase in density as in muscle atrophy was observed with cachexia compared to the control group. Neither cachexiaassociated upregulation of muscle-specific E3-ligases TRIM63 and FBXO32 of the ubiquitin system, commonly used as proteolysis markers, nor upregulation of BAX and CASP3 (proapoptotic signals) were detected. But the protein expression of Fas ligand was 2.32-fold increased and the expression of RNA of the antiapoptotic BCL2 was significantly 0,49-fold lower (p=0.028). Abundance of cells with centralized nuclei was significantly increased by 7% (p=0.031) in muscle fiber type I and over all fiber types by 5% (p=0.085), likely indicating increased muscle regeneration, though PAX7 expression remained unaltered. As a main new finding, there was a significant 20% (p=0.022) decrease in the intracellular concentration of the proteinanabolic and antiproteolytic amino acid leucine, which, notably, correlated to weight loss (p=0.046). The concomitant finding of 1.782- and 1.822-fold increased expression of the transmembrane transporters SNAT2 (p=0.013) and LAT1 (p=0.072), respectively, did not allow any conclusion on (limited) leucine uptake, especially as no blood samples were available for extracellular leucine measurements. The present study provided evidence for a pro-inflammatory milieu with cachexia compared to controls in terms of a significantly increased expression of CD68 (1.48-fold, p=0.040), indicating macrophage infiltration, and, moreover, a significantly decreased relative expression of GCS (0.831-fold; p=0.036) and GSR (0.657-fold, p=0.026) as involved in syntheses and reduction of the antioxidant glutathione, respectively. Correspondingly the following inflammatory factors were increased in the protein array: IL1β (1.41-fold), IL6 (1.50-fold), IL8 (1.48-fold), TNF (1.87- fold), interferon-y (1.14x), CRP (1.55-fold), and IL32 (3.03-fold). The mRNA expressions of IL1β, IL6, IL8 and TNF were, however, not significantly increased. The mRNA expressions of the monoamine oxidases, as a possible source of oxidative stress, were inconsistent, as changes in MAOA expression were 0.19-fold (p=0.030) while those of MAOB were 1.4-fold (p=0.047). In line with pro-inflammatory changes, a 33% increase in capillary contacts of the muscle fibers (p=0.097) was observed along with, by trend, higher protein levels of pro-angiogenetic signals: VEGF was 1.30-fold, angipoietin-1 2.07-fold, Angiopoietin-2 1.77-fold increased and PF4 was 0.16-fold suppressed. The mRNA expressions of VEGFA, VEGFB and KDR were, however, unchanged. In synopsis of these new human data on human skeletal muscle from patients with pancreatic cancer, it can be stated that at an early pre-atrophic stage of cachexia intracellular leucine, as an essential anabolic mediator, is significantly reduced. At the same time there is an inflammatory milieu associated with a trend towards angioneogenesis and possibly increased muscle regeneration. This early metabolic and inflammatory condition in not yet atrophied skeletal muscle may be relevant as a possible target for prevention and a therapeutic approach to cancer cachexia. A relevant time window and a set of parameters for characterization of a “myositis-like-phase” of cachexia development were identified for further clinical studies.