DNA methylation and gene expression characterization in peripheral blood mononuclear cells of lung cancer patients with or without anorexia: a case-control study with high throughput approach

Anorexia, i.e., the loss of appetite and adipose and muscle tissue wasting, is a hallmark of several types of cancer, including lung cancer. Cancer-associated anorexia is a multifactorial disabling symptom that negatively characterizes patients’ prognosis, whose pathogenic mechanisms still remain unclear. The main aim of this project was to characterize gene expression and DNA methylation profiles in peripheral blood mononuclear cells (PBMCs) of lung cancer patients with or without anorexia. PBMCs were chosen to identify possible clinically useful biomarkers in an easily accessible human tissue. Transcriptome profile was assessed by RNA sequencing in anorexic cancer patients, non-anorexic cancer patients and healthy controls, and validated by real time quantitative PCR (RT-qPCR). Gene expression data showed the most relevant differences in the comparisons of cancer patients with controls and anorexic cancer patients with controls. Differentially expressed genes were involved in immune regulation pathways, such as inflammation signaling, chemokine and cytokine-mediated inflammation signaling, TGFbeta and interleukin signaling. RT-qPCR analysis was used to validate RNA sequencing data, in particular ADAM8 and SMAD4 were down-regulated in cancer patients and anorexic cancer patients compared to controls and CLU and CCR4 were up-regulated in anorexic cancer patients compared to non-anorexic cancer patients. DNA methylation profiles were assessed with microarray-based genome-wide methylation analysis and then validated by targeted bisulfite sequencing. Genome-wide methylation data were integrated with RNA sequencing data to identify genes that were differentially methylated and simultaneously differentially expressed. Four genes (GNL3L, FHL1, FLNA and PGRMC1) that resulted hypomethylated induced in anorexic cancer patients compared to controls, were selected to validate the methylation status. Targeted bisulfite sequencing confirmed genome-wide data and interestingly showed a progressive demethylation from non-anorexic cancer patients to anorexic cancer patients as compared to controls. According to our findings, we could speculate about a possible relationship of lung cancer-associated anorexia with gene expression changes and DNA methylation alterations in PBMCs, although further analyses are necessary to confirm these data and better understand the mechanisms underlying cancer-associated anorexia.