Profiling and Identification of Post-Transcriptional Regulatory Molecules for Human Cancer Cachexia

  • Author / Creator
    Narasimhan, Ashok
  • Cancer cachexia (CC) is a multifactorial syndrome characterized by severe depletion of skeletal muscle, with or without fat loss. A majority of cancer patients with incurable cancer are affected with cachexia and it remains an unmet medical need. Gene expression changes at the transcriptional level have been studied for CC in both humans and rodents. However, post-transcriptional regulatory mechanisms likely play a role in CC and are not addressed in the literature on human subjects, and are the focus of this thesis. It is well known that the sex-specific expression of genes contributes to traits and phenotypes. The role of autosomal expression of genes and their regulation at a post-transcriptional level in skeletal muscle from cancer cachexia patients has not been explored to account for the sex-specific differences in cachexia prevalence or severity. I hypothesize that CC, a manifestation of the net effect of complex host-tumor interactions and underlying molecular mechanisms, involves dysregulation of miRNAs and splice variants in skeletal muscle contributing to the pathophysiology of CC in a sex-specific manner. The objectives of the thesis are (i) to profile miRNAs expressed in skeletal muscle biopsies and to identify Differentially Expressed (DE) miRNAs associated with human CC using Next Generation Sequencing (NGS), (ii) to profile Alternatively Spliced Genes (ASGs) from human skeletal muscle biopsies and to identify differentially expressed alternatively spliced genes (DASGs) associated with human CC using Human Transcriptome Array 2.0 (HTA 2.0) and (iii) to profile and identify sex-specific miRNAs and Differentially Expressed Isoform specific gene expression (DEI) for human CC. Study subjects were classified based on the international consensus definition and diagnostic criteria for CC. Forty-two cancer patients were classified into 22 cachectic cases and 20 non-cachectic cancer controls who had no weight loss over a period of six months compared to their pre-illness weight. For identifying sex-specific expression differences, 22 cases and 20 controls were further stratified by sex. Body composition analysis was carried out using computed tomography (CT) taken prior to surgery. Total RNA was isolated from rectus abdominis muscle and used for NGS and HTA 2.0 arrays. Representative signatures were validated using qRT-PCR for cross platform concordance. Partek Genomics Suite v6.6 and SPSS v16 were used for bioinformatics and statistical analyses, respectively. Ingenuity pathway analysis (IPA) was used to identify canonical pathways and network analysis for biological interpretation. 777 miRNAs were profiled and 82 miRNAs with read counts of ≥ 5 in 80% of samples were retained for analysis. Eight miRNAs were DE (up-regulated, fold change of ≥ 1.4 at p < 0.05). IPA identified pathways related to myogenesis and inflammation. qRT-PCR analysis of representative miRs showed a similar direction of effect (p < 0.05), as observed in NGS. 8960 ASGs were identified, of which 922 DASGs (772 up-regulated, 150 down-regulated) were observed with > 1.4 fold change and p < 0.05. Representative DASGs validated by semi-quantitative RT-PCR confirmed the primary findings from the HTA 2.0. Identified DASGs were associated with myogenesis, adipogenesis, protein ubiquitination and inflammation. Up to 10% of the DASGs exhibited cassette exon (exon included or skipped) as a predominant form of AS event. Other forms of AS events such as intron retention and alternative promoters were also observed. Sex-specific expression was observed both at the miRNA and at the DEI levels. 10 upregulated miRNAs in males and 3 upregulated miRNAs in females were identified with ≥ 1.4 FC and p < 0.05. 1324 and 372 DEIs were identified in males and females, respectively. Pathways associated with skeletal muscle atrophy were predominant in males, whereas adipogenesis pathways were more common in females. Although the same upstream regulators were identified, their downstream targets were different between sexes. In all, my study has shown for the first time that post-transcriptional regulatory molecules are indeed associated with the pathophysiology of CC and are expressed in a sex-specific manner. This may potentially spur interests to address finer molecular mechanisms as an approach to personalized therapeutics development based on sex. Independent replications of the study findings are warranted. Functional characterization of the identified molecules may offer insights into their biological significance in cancer cachexia.

  • Subjects / Keywords
  • Graduation date
    2017-06:Spring 2017
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Medical Sciences-Laboratory Medicine and Pathology
  • Supervisor / co-supervisor and their department(s)
    • Damaraju, Sambasivarao (Laboratory Medicine and Pathology)
    • Baracos, Vickie (Oncology)
  • Examining committee members and their departments
    • Greiner, Russell (Computing Science)
    • Yokota, Toshifumi (Medical Genetics)
    • Zimmers, Teresa (Surgery, Indiana University)