Real-imaging cDNA-AFLP transcript profiling of pancreatic cancer patients: Egr-1 as a potential key regulator of muscle cachexia

Abstract

Background: Cancer cachexia is a progressive wasting syndrome and the most prevalent characteristic of cancer in patients with advanced pancreatic adenocarcinoma. We hypothesize that genes expressed in wasted skeletal muscle of pancreatic cancer patients may determine the initiation and severity of cachexia syndrome.

Experimental design: We studied gene expression in skeletal muscle biopsies from pancreatic cancer patients with and without cachexia utilizing Real-Imaging cDNA-AFLP-based transcript profiling for genome-wide expression analysis.

Results: Our approach yielded 183 cachexia-associated genes. Ontology analysis revealed characteristic changes for a number of genes involved in muscle contraction, actin cytoskeleton rearrangement, protein degradation, tissue hypoxia, immediate early response and acute-phase response.

Conclusions: We demonstrate that Real-Imaging cDNA-AFLP analysis is a robust method for high-throughput gene expression studies of cancer cachexia syndrome in patients with pancreatic cancer. According to quantitative RT-PCR validation, the expression levels of genes encoding the acute-phase proteins α-antitrypsin and fibrinogen α and the immediate early response genes Egr-1 and IER-5 were significantly elevated in the skeletal muscle of wasted patients. By immunohistochemical and Western immunoblotting analysis it was shown, that Egr-1 expression is significantly increased in patients with cachexia and cancer. This provides new evidence that chronic activation of systemic inflammatory response might be a common and unifying factor of muscle cachexia.

Figure 1

UICC Classification of the 23 patients operated for pancreatic cancer. Distribution of tumor stage of all pancreatic cancer patients was determined according to the 5th edition of the TNM classification of the Union International Contre Cancer (UICC). Median weight loss of the cachectic patients was 13.9% of initial body weight within 6 months comparing with the patients without cachexia in which the median weight loss was 2.1% respectively. The resection rate was lower in patients with cachexia (50% vs. 92.3%) UICC II: without distant metastases; UICC III: infiltration of celiac trunk and/or mesenteric artery; UICC IV: metastases in liver and/or peritoneal cavity according to guidelines UICC 2002) in patients either with (n = 10) or without cachexia (n = 13).

Figure 2

cDNA-AFLP Transcript Profiling of pancreatic cancer patients. cDNA-AFLP tag patterns amplified from mRNA of skeletal muscle biopsy samples of pancreatic cancer patients either with cachexia (IRD800-labeled, red bands) or without cachexia (IRD700-labeled, green bands) were loaded in the same lane according to their primer combinations and separated in a 8% denaturing polyacrylamide gel. Data were collected on automated Infrared Sequencer DNAIR 4200 (Li-COR Biosciences GmbH). Three different cDNA-AFLP tag profiles (i), (ii) and (iii) of wasted patients (red bands) were compared with one non-cachectic control (green bands).

Figure 3

Preparative Real-Imaging cDNA-AFLP Screening.A) cDNA tag patterns were amplified with individual primer combinations Taq+2 and Mse+2 (Taq+2 was labeled with IRD700). After electrophoresis, the gel was transferred and dried on green grid paper and scanned using Odyssey Imaging System (Li-COR Biosciences GmbH). The lanes are occupied by cachexia-associated samples (+) and non-cachectic probes (−) allowing direct visual comparison. Grid coordinates are indicated in millimeters (mm). B) Differentially expressed tag patterns (marked in red boxes) were isolated for sequencing analysis.

Figure 4

Altered expression of Egr-1 in the skeletal muscle tissue of wasted pancreatic cancer patients. Immunohistochemical staining of skeletal muscle tissue shows significantly enhanced expression of Egr-1 in wasted pancreatic cancer patients (C, D) compared with non-cachectic patients (A, B). A, C – longitudinal sections; B, D – transverse sections of skeletal muscle from pancreatic cancer patients. 40X original magnification.

Figure 5

Egr-1 protein is markedly induced in skeletal muscle of wasted pancreatic cancer patients. Protein levels of skeletal muscle EGR-1 and β-tubulin (loading control) were determined by Western immunoblotting showing aberrant upregulation of Egr-1 in skeletal muscle of patients with cancer cachexia. Western immunoblotting of Egr-1 (green band, 82 kDa) and β-tubulin (red band, 55 kDa) compares (A) two non-cachectic with two cachexia-associated biopsy protein extracts and (B) one non-cachectic probe with three cachexia-associated skeletal muscle biopsy samples. Similar results were obtained in three independent experiments. M – coomassie-blue prestained protein marker. Full-length blots/gels are presented in Additional file 3: Figure S2. (C) Densitometric analysis of Egr-1 protein expression. Median rate = 10.23, P-value ≤ 0.005 according to Student’s t-test.