Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Dr. Lynne-Marie Postovit

Abstract

Nodal and related ligands are highly conserved members of the TGF-beta superfamily with well-established and essential roles in the early embryonic development of vertebrates, and in cell fate decisions in human embryonic stem (hES) cells. Aberrant NODAL signaling also generally promotes pro-tumourigenic phenotypes and the progression of a wide array of human cancers. Despite being pursued as a potential therapeutic target, many aspects of NODAL’s molecular biology remain poorly understood. This thesis provides a comprehensive characterization of gene expression from the human NODAL locus at multiple levels. First, an intronic NODAL SNP known as rs2231947 was found to be functional in its modulation of a novel alternatively spliced exon. This exon contributed to a full-length processed NODAL variant transcript. The existence of this genetically regulated NODAL isoform suggests that NODAL biology is more complex than currently appreciated. At the protein level, the alternatively spliced NODAL variant differs in the C-terminal half of the NODAL mature peptide. The NODAL variant was preferentially secreted relative to constitutive NODAL, but displayed similar extracellular stability and processing. Differential N-glycosylation was partially responsible for this increased secretion, and for NODAL secretion in general. The NODAL variant protein is unlikely to adopt a constitutive NODAL-like structure, and did not induce expression of targets of canonical NODAL signaling in the zebrafish embryo. However, the NODAL variant did efficiently complex via inter-chain disulfide bonds, and induced pro-tumourigenic phenotypes to a limited extent relative to constitutive NODAL. In summary, this work demonstrates previously unknown complexity governing human NODAL gene expression and function. These molecular details will help broaden our understanding of NODAL function as well as aid in the continued development of potential targeted therapies to inhibit NODAL signaling in cancer.

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