Function of Plasma Membrane and a3-containing V-ATPases in Breast Cancer.
Cotter, Kristina.
2016
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Abstract:
Metastasis, or the spread of tumor cells from a primary site to secondary sites in the
body, is the leading cause of cancer mortality. The development of therapeutics that
target key components of this cascade is thus of critical importance. Metastasis
requires that cells migrate and invade through extracellular matrix. The vacuolar
(H+)-ATPases (V-ATPases) are ubiquitously expressed, ... read moreATP-dependent proton pumps that
have been implicated in invasion and migration. V-ATPases acidify intracellular
compartments as well as the extracellular space of specialized cells, and consist of a
peripheral V1 domain and a membrane-embedded V0 domain. Reversible assembly of these
domains is a key mechanism to regulate activity. Elucidation of pump structure,
regulation, and function in tumor cells is critical to determine whether it could serve
as a drug target. This thesis offers insight into the V-ATPase as a novel therapeutic
target to limit metastasis. Subunit a of the V0 domain consists of a C-terminal domain
involved in proton translocation and an N-terminal domain involved in assembly and
V-ATPase localization to specific cellular membranes. Despite its importance, there is
no high-resolution structure for subunit a. We have used homology modeling to construct
a model of the N-terminal domain of Vph1p, a yeast subunit a isoform. This model was
tested by assessing the accessibility of unique cysteine residues to chemical
modification. This method has allowed for identification of regions of subunit a that
likely interface with V1. The results from this study offer insight into how we may
develop isoform-specific inhibitors. In yeast, assembly is regulated by the Ras/cAMP/PKA
pathway downstream of glucose. The mechanisms by which PKA modulates pump assembly is
not known. We have performed an EMS mutagenesis screen to identify regulators of
V-ATPase assembly downstream or independent of PKA in yeast. Our screening method
isolated several mutants that are potentially defective in glucose-dependent
disassembly. Future work will identify the mutated genes that yield this phenotype and
enhance our understanding of how V-ATPase activity may be modulated in both yeast and
higher eukaryotes. The V-ATPase is present on the plasma membranes of at least some
invasive cancer cells and inhibition of V-ATPases reduces cancer cell invasion. It has
thus been proposed that plasma membrane pumps are critical for invasion. To test this,
we employed both an antibody and small molecule approach to specifically ablate plasma
membrane V-ATPase activity. Inhibition of these pumps significantly reduces invasion and
migration of breast cancer cells in vitro. In mammals, V-ATPase localization is
controlled by isoforms of subunit a (a1-a4). Previous work from our lab has shown that
the a3 isoform is overexpressed in a number of highly invasive breast cancer cell lines
and reduction of its expression reduces in vitro invasion. Using an a3-specific
antibody, we found that a3 localizes to the leading edge of migrating breast cancer
cells and, using a3-specific siRNA knockdown, have shown that a3 is critical for
migration of breast cancer cells. Importantly, using quantitative RT-PCR, we have found
that a3 is overexpressed in all 43 human breast cancer samples tested relative to normal
breast tissue. Moreover, using immunofluorescence microscopy, we have shown that a3 is
expressed at higher levels in invasive breast carcinoma relative to solid tumors and
normal breast tissue. The results from these studies suggest that a3-containing plasma
membrane V-ATPases may represent a novel and important anti-metastatic target. An
important question is how V-ATPases might contribute to breast cancer metastasis. We
have begun to explore the hypothesis that V-ATPases at the plasma membrane of breast
cancer cells provide an acidic extracellular environment that aids in the proteolytic
activation and catalytic activity of pH-dependent proteases such as cathepsins. These
proteases may in turn promote invasion by digestion of extracellular matrix and
activation of other proteases. Future studies will explore both the mechanism by which
V-ATPases promote tumor cell invasion and the role of plasma membrane and a3-containing
V-ATPases in metastasis in vivo.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Cellular & Molecular Physiology.
Advisors: Michael Forgac, and Peter Juo.
Committee: Brent Cochran, Phil Hinds, Laura Liscum, and Patricia Kane.
Keyword: Physiology.read less - ID:
- 028717465
- Component ID:
- tufts:20328
- To Cite:
- TARC Citation Guide EndNote
