UVM Theses and Dissertations
Format:
Print
Author:
Bradbury, C. Matthew
Dept./Program:
Cell and Molecular Biology Program
Year:
2008
Degree:
PhD
Abstract:
Melanoma is the most lethal form of skin cancer. It is characterized by a rising incidence in the American population and is potentially lethal in patients with advanced disease. High mortality is associated with disseminated metastases and inadequate systemic therapies, which overwhelmingly fail. This study is rooted in the premise that more thorough understanding of melanoma biology, highlighting differences of tumor cell biology from melanocyte precursors, may provide insights necessary to develop novel therapeutics for metastatic melanoma. Dysregulated gene expression is a hallmark of oncogenesis, and previous investigations by our research group have attempted to examine the contribution of gene expression changes to melanoma formation. A series of genes have been identified as candidate tumor suppressors for their consistent and substantial loss of expression in ten established human melanoma cell lines relative to primary human foreskin melanocytes. Two of these genes, CCL3 and PARC, have been previously characterized as chemokines. Chemokines, in general, have been implicated in cell transformation, tumor cell survival, tumor-host interactions, invasion, and metastasis.
By convention, chemokines signal through a discrete series of G-protein coupled receptors at the cell surface. CCL3 and PARC, in particular, have been described to adopt critical roles in chemotaxis, hematopoietic stem cell regulation, and immune modulation. However, they are distinct proteins with modes of regulation, genetic sequences, signaling mechanisms, and functional consequences separate from each other. Furthermore, PARC, with its more recent evolutionary development, has no homolog outside of hominids, and no signaling receptor has been identified. Importantly, no tumor suppressive function has been characterized for either of these genes in any cancer model system. To elucidate roles for CCL3 and PARC as novel tumor suppressors in melanoma, cDNA constructs containing full-length genes of interest were generated and stably transfected into the MelJuSo human melanoma cell line. Assays established diminished in vivo and in vitro growth potential for melanoma cell lines expressing CCL3 and PARC, relative to vector-transfected controls. These findings demonstrate a potentially novel tumor suppressor function for each protein. Restoration of CCL3 or PARC decreased in vitro colony-forming capacity, resulted in increased sub-populations of cells in GoIGl cell cycle phase and a larger fraction of melanoma cells undergoing apoptosis. Attempts to recapitulate growth inhibitory effects by treating parental melanoma cells with exogenous recombinant human CCL3 or PARC bioactive ligands did not cause tumor suppression.
However, increased levels of endoplasmic stress-related proteins in CCL3- and PARC-expressing melanoma cells implicate the unfolded protein response as a mechanism of cell death. While unexpected, these results are not unprecedented mechanisms of tumor suppression by endogenously expressed chemokines. Mounting evidence suggests that ligands can adopt divergent mechanism when acting from extracellular sources than the potential tumor suppressive functions when acting intracellularly. The present study contributes significantly to the growing body of work suggesting dichotomous roles for chemokines in tumor cellular physiology, and implementation of either of these molecules as therapeutics must take these roles into account.
By convention, chemokines signal through a discrete series of G-protein coupled receptors at the cell surface. CCL3 and PARC, in particular, have been described to adopt critical roles in chemotaxis, hematopoietic stem cell regulation, and immune modulation. However, they are distinct proteins with modes of regulation, genetic sequences, signaling mechanisms, and functional consequences separate from each other. Furthermore, PARC, with its more recent evolutionary development, has no homolog outside of hominids, and no signaling receptor has been identified. Importantly, no tumor suppressive function has been characterized for either of these genes in any cancer model system. To elucidate roles for CCL3 and PARC as novel tumor suppressors in melanoma, cDNA constructs containing full-length genes of interest were generated and stably transfected into the MelJuSo human melanoma cell line. Assays established diminished in vivo and in vitro growth potential for melanoma cell lines expressing CCL3 and PARC, relative to vector-transfected controls. These findings demonstrate a potentially novel tumor suppressor function for each protein. Restoration of CCL3 or PARC decreased in vitro colony-forming capacity, resulted in increased sub-populations of cells in GoIGl cell cycle phase and a larger fraction of melanoma cells undergoing apoptosis. Attempts to recapitulate growth inhibitory effects by treating parental melanoma cells with exogenous recombinant human CCL3 or PARC bioactive ligands did not cause tumor suppression.
However, increased levels of endoplasmic stress-related proteins in CCL3- and PARC-expressing melanoma cells implicate the unfolded protein response as a mechanism of cell death. While unexpected, these results are not unprecedented mechanisms of tumor suppression by endogenously expressed chemokines. Mounting evidence suggests that ligands can adopt divergent mechanism when acting from extracellular sources than the potential tumor suppressive functions when acting intracellularly. The present study contributes significantly to the growing body of work suggesting dichotomous roles for chemokines in tumor cellular physiology, and implementation of either of these molecules as therapeutics must take these roles into account.