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Amal Hussain Ibrahim Al Haddad
Department of Physiology
College of Medicine and Health Sciences
Dissertation
Title
Identifying the Molecular Mechanisms of Early Cachexia Using Whole Transcriptome
Sequencing in Muscle and Fat Biopsies from Cancer Patients
Faculty Advisor
Prof. Thomas E. Adrian
Defense Date
10 June 2015
Abstract
Cachexia causes one third of cancer–related deaths and contributes to that of many others. Despite extensive
research, themechanisms of cancer cachexia are poorly understood. Identification of early changes in gene expression
in the major cachexia target tissues will improve the understanding of its mechanisms. We investigated the entire
transcriptome, using next generation sequencing (Illumina HiSeq 2500), to identify altered expression of genes in muscle
and fat from cancer patients. Samples of rectus abdominis muscle and visceral fat were collected at surgery from
patients exhibiting 5-10% weight loss prior to surgery, compared with stable-weight patients. Also, selected differentially
ex-pressed genes were confirmed using real-time RT-PCR. In muscle, 30 genes showed highly significant change in
expression (25 down and 5 up: P<0.0005 - P<0.00001, FDR 0.2). The 25 downregulated genes included 7 involved with
metabolism (5 mitochondrial); 4 with signaling; 4 with ubiquitination; and 3 with intracellular trafficking. Multiple genes
involved in glycogen metabolism were downregulat-ed, correlating with the lack of glycogen, muscle weakness, and
fatigue; characteristic of cachexia. The 5 upregulated genes include 2 involved with calcium signaling and 2 with cell
matrix interactions. Expres-sion of genes previously thought to be important in cachexia, including several inflammatory
cyto-kines, was not significantly different. FBXO32, which encodes atrogin-1, upregulated in an in vitro cachexia model,
was actually downregulated. No transcripts for the dermicidin gene, which codes for proteolysis-inducing factor, were
detected. Expression of myostatin and its receptor (ACTR2B) were significantly decreased, possibly reflecting end
organ adaptation to tumor produced myostatin. In visceral fat, expression of 6 genes were downregulated and 10
upregulated with high statistical signifi-cance (P<0.001-0.0002). Several of these encode metabolic enzymes. Of genes
in fat previously impli-cated with cachexia, such as hormone sensitive lipase and adipose tissue triglyceride lipase,
were un-changed. In contrast, leptin was significantly downregulated and the zinc-α-2-glycoprotein (lipid mobi-lizing
factor) was significantly upregulated as expected. These studies explain some documented evi-dence in cachexia
pathogenesis, highlight ambiguous data from animal models, and reveal unexpected changes in gene expression
that underlie the pathophysiology of the cachectic state in cancer. These results bring reliable, representable, and
consistent data from the clinic and back to the bench with more focused insights to be investigated and verified.
Research Relevance and Potential Impact
Most patients with cancer exhibit marked wasting of skeletal muscle and anorexia which is known as the cancer cachexia syndrome. Cachexia
is a major cause of cancer death and it affects the quality of life, the response to therapy, the ability to withstand the rigors of therapy and even
the psychological wellbeing of the patients and their families. The mechanisms of cancer cachexia are not well understood. We hypothesized
that changes in gene expression in early cachexia would shed light on pathways involved which would then pave the way for a more targeted
approach to therapeutic intervention. The entire transcriptome of skeletal muscle and visceral adipose tissue was investigated using next-generation
sequencing and the major changes confirmed by fast real-time RT-PCR. These studies indicate the involvement of several metabolic pathways
that explain much of the clinical observations in cancer cachexia. The data confirms some of the documented evidence in the pathogenesis of
cachexia, highlights ambiguous data from animal models, and reveals unexpected changes in gene expression that underlie the pathophysiology
of the cachectic state in cancer. Once confirmed in other groups of patients with cachexia these findings are likely to lead to improvements in the
prevention and treatment of cachexia in patients with cancer.