Cachexia is present in around 80% of patients with cancer in advanced stages, inducing marked depletion of lean and fat mass, and in more than 20% of patients cachexia is responsible for death . Whereas starvation and chronic malnutrition are associated with adaptive decreases in metabolic rate, cancer cachexia is characterized by altered metabolic processes and activation of catabolic pathways .
We hypothesized that alteration in gene expression within the skeletal muscle in response to advanced pancreatic cancer plays a crucial role in the development of the muscle cachexia syndrome. To test this hypothesis, we carried out the transcriptome-wide characterization of skeletal muscle biopsies utilizing the Real-Imaging cDNA-AFLP approach to identify those genes that are commonly induced in the skeletal muscle of pancreatic cancer patients either with or without muscle cachexia.
In contrast to other studies of skeletal muscle cancer cachexia based on cDNA microarray technique[14, 15], our Real-Imaging cDNA-AFLP approach gives the ability to identify and annotate expressed genes without prior sequence knowledge making possible to distinguish among different transcripts from genes belonging to the same gene family or splice variants of the same gene. Importantly, our results indicate that molecular signatures of gene expression appear to be predictive of muscle cachexia, before we can measure this in the preoperative collected blood samples as there is no significant difference in protein levels or CrP-levels to detect. We suggest that in characterizing the skeletal muscle transcriptome, one might expect that different signal systems may work in combination to shift the disease stage of pancreatic cancer patients towards cachectic conditions.
We observed that transcript levels of contractile proteins in skeletal muscle of patients with cachexia change in the same direction. We observed more than 6-fold upregulation of cyclin-dependent kinase inhibitor p21 (Table 3). The mRNA levels of increased p21 mRNA expression in cachectic patients may reflect increased activity of myostatin, a negative regulator of muscle growth and development which is thought to induce cachexia . The skeletal muscle mass appears to be significantly involved in the process of cancer cachexia in patients with pancreatic cancer, being the potential target of various tumor products which can activate protein degradation . All these changes may indicate the abnormal metabolic processes shifting towards hypermetabolism and negative energy expenditure in patients with cachexia.
We have also observed the induction of genes encoding basic helix-loop-helix (bHLH) transcription factors HES1, DEC1, and DEC2, which are primarily associated with hypoxia (Table 3). As it was reported previously, hypoxia enhances Notch signaling and activates Notch-responsive promoters and increases expression of bHLH transcription factors . HES1 and DEC1 (Table 3) are immediate downstream targets of Notch signaling inhibiting the transcriptional expression and/or function of lineage-specifying genes involved in myogenesis .
Our study demonstrates for the first time that the genes encoding acute-phase response (APR) proteins fibrinogen α, α1-antitrypsin and α2-macroglobulin are markedly upregulated not only in the liver but also in the skeletal muscle of wasted patients with pancreatic cancer (Table 3), which are associated with altered energy expenditure and increased protein metabolism of pancreatic cancer patients . It has been shown that approximately 40 percent of pancreatic cancer patients show an acute-phase response at the time of diagnosis, and this amount increases to around 80 percent at the time of death .
While acute-phase response indicates systemic inflammation, the pro-inflammatory cytokines appear to mediate this response. Furthermore, it has been reported that non-immune tissues such as cardiac and skeletal muscle appears to express pro-inflammatory cytokines . In excellent agreement with these facts, we observed 6-fold upregulation of interleukin IL-32 (Table 3), play an important role in inflammatory diseases as direct inducer of TNF-α .
Notably, in contrast to the previous study on human skeletal muscle cancer cachexia , we observed a more than 5-fold upregulation of FOXO1, a key regulator of gene expression during skeletal muscle atrophy  (p<0.05, Table 3). We suppose that it may be explained by more stringent selection of cachectic patients in our study with weigh loss criteria from 10 to 19% within 6 months (Table 1).
Importantly, together with an ongoing inflammatory response we observed the significant induction of early response genes Egr-1 and IER-5 in wasted pancreatic cancer patients (p<0.05, Table 3). Conceptually, Egr-1, the master switch regulating inflammatory parameters, can be viewed as a main initiator of inflammatory response, as it is directly involved in “early signal events”[24, 25]. Egr-1 is also recognized as playing a critical role in the regulation of over 40 target genes, including tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, all of which can induce various features of cachexia syndrome in the skeletal muscle in animal models . Together with elevated acute phase response gene expression, the Egr-1 may serve as a biomarker of muscle cachexia syndrome.