Publication Date
2012
Document Type
Thesis
Committee Members
Gerald Alter (Committee Member), Steven Berberich (Committee Member), Julian Gomez-cambronero (Committee Chair)
Degree Name
Master of Science (MS)
Abstract
Phospholipase D (PLD) is an enzyme that breaks down phospholipids in the cell membrane. It has been suggested that PLD may play a role during cell proliferation and cell invasion of cancer cells. The objective of this thesis was to define new molecular signaling pathways in which PLD2 might be involved in terms of cell proliferation (first part) and cell invasion (second part). To this, I compared molecular and biochemical aspects between untransformed cell lines with highly invasive, transformed breast cancer cells. In the first part, I investigated the interaction of two tyrosine kinases with PLD2 and the effect of such interaction on cell proliferation in the highly invasive MDA-MB-231 breast cancer cells. I found that the activities from the two kinases and from PLD2 are all augmented in the cancer cells while the protein levels are only relatively high in these cells. JAK3 upregulates PLD2 activity in MDA-MB-231 cells while inhibits PLD2 in the untransformed breast epithelial MCF10A cells. Fes interacts with PLD2 at distinct binding sites in MDA-MB-231 cells compared to MCF10A cells. I also found that phosphatidic acid (PA), the product of PLD enzymatic activity, enhances Fes activity in MDAMB-231 cells but not in MCF10A cells. Further, I demonstrate that the two tyrosine kinases JAK3 and Fes also interact with each other, with Fes having an inhibitory effect on JAK3 in MCF10A cells but not in cancer cells. The balanced PLD2 activity by JAK3, Fes and PLD2 interactions in the MCF10A cells keeps PLD2 at basal level that lead to normal cell growth. In contrast, in MDA-MB-231 cancer cells, the effect of positive feedback among the three proteins and then enhancing effect of PA contribute to augmented proliferation.
In the second part of this thesis, I investigated the role of JAK3 on PLD2 activities during cell invasion in MDA-MB-231 cells using a small-molecule tyrosine kinase inhibitor, the flavonoid apigenin (4',5,7-trihydroxyflavone), as well as RNA silencing. I found that that both JAK3 and the tyrosine kinase, Epidermal Growth Factor Receptor (EGFR) are sensitive to the inhibitors and directly regulate PLD2 and induce the highly invasive phenotype of MDA-MB-231 cells. Further, serum-deprived cells in culture show an upregulated EGFR/JAK3/PLD2-PA system and are especially sensitive to a combination of JAK3 and PLD2 enzymatic activity inhibitors (i.e. 30 nM apigenin and 300 nM 5-Fluoro-2-Indolyl des-Chlorohalopemide [FIPI], respectively). Accordingly, cell invasion is enhanced by two kinases (EGFR and JAK3) and a phospholipase (PLD2) on multiple levels that provides regulatory flexibility and contribute to maximum aggressiveness in cell invasion in MDA-MB-231 breast cancer cells. This is especially important during serum starvation that coincides with migration of these cells to new locations.
In this thesis, I have demonstrated the existence of new signaling pathways: JAK3-Fes-PLD2 that plays a central role in cancer cell proliferation and EGFR-JAK3-PLD2 that facilitate cell invasion. These new pathways might be important to be used as therapeutic targets aimed at controlling the high proliferative and invasive phenotype of breast cancer cells.
Page Count
90
Department or Program
Department of Biochemistry and Molecular Biology
Year Degree Awarded
2012
Copyright
Copyright 2012, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.
