Regulation of V-ATPase Activity and its Role in Breast Cancer Metastasis
McGuire, Christina.
2019
-
The vacuolar H+
ATPase (V-ATPase) is an ATP driven proton pump made up of fourteen different subunits
that are arranged into two domains, the peripheral V1 domain and the integral V0 domain.
Present in both intracellular membranes and at the plasma membrane of specialized cell
types, V-ATPase activity is required for numerous basic cellular processes.
Dysregulation of V-ATPase activity has ... read morebeen implicated in various diseases including
cancer, renal tubular acidosis, and osteopetrosis. One of the major ways pump activity
is controlled, is through the reversible assembly of its two domains. The process of
reversible assembly is conserved across eukaryotes ranging from yeast to mammals, though
the signaling pathways controlling it are not fully understood. Here, we identify
glucose starvation as a novel regulator of V-ATPase assembly in mammalian cells. Acute
glucose starvation induced a rapid and reversible increase in both assembly and activity
of the V-ATPase. Because the V-ATPase is required for the activation of AMP Kinase
(AMPK), a critical cellular energy sensor that is also activated upon glucose
starvation, we hypothesized that V-ATPase assembly regulated AMPK activation. To test
this, we compared the time course of AMPK activation and V-ATPase activity upon glucose
starvation. We observed that activation of AMPK preceded an increase in pump activity,
suggesting that regulated assembly was not contributing to AMPK activation during
glucose starvation. Moreover, a pharmacological AMPK inhibitor prevented the
starvation-induced increase in V-ATPase activity and assembly. These results suggested
that increased assembly and activity of the V-ATPase during glucose starvation are due
to AMPK signaling. Surprisingly, after performing a genetic knockout of AMPK we still
observed an increase in pump activity after glucose starvation, indicating that our AMPK
inhibitor was targeting a molecule other than AMPK. Using additional pharmacological
inhibitors of important signaling pathways within the cell we found that the PI3K/Akt
pathway, which has previously been implicated in controlling V-ATPase assembly in
mammalian cells, plays a role in the starvation-induced increase in V-ATPase assembly
and activity. In addition to functioning in cellular homeostasis, V-ATPase activity also
promotes cancer cell survival and metastasis. Previous work in our lab identified plasma
membrane V-ATPases as key players in breast cancer cell invasiveness. The two subunit-a
isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, where
expression of a3 promotes plasma membrane pump expression, and thereby invasiveness, of
human invasive breast cancer cell lines. We sought to analyze the role of each of the
subunit a-isoforms in the invasive, 4T1-12B mouse breast cancer cell line. Based on work
in human breast cancer cells, we hypothesized that a3 would be the dominant a-isoform
that also promoted the invasive phenotype of these cells. Surprisingly, we found that a4
was the dominantly expressed a-isoform in these cells. Genetic knockout of each of the
four a-isoforms using CRISPR/Cas9 revealed that the a4 isoform promoted plasma membrane
expression of the V-ATPase in 4T1-12B cells, and knockout of this isoform, but not
isoforms a1-a3, reduced cell migration and invasion. These findings suggest that while
different a-isoforms may upregulate plasma membrane V-ATPase localization, cell surface
pumps promote an invasive phenotype. This work identifies glucose starvation as a novel
regulator of V-ATPase assembly during glucose starvation in mammalian cells and attempts
to understand the mechanism through which V-ATPases contribute to AMPK activation.
Furthermore, it identifies subunit a4 as the dominant a-isoform in 4T1-12B cells, that
also localizes the V-ATPase to the cell surface to promote invasiveness. Together, this
provides novel insights into both the mechanisms of cellular homeostasis and cancer cell
metastasis.
Thesis (Ph.D.)--Tufts University, 2019.
Submitted to the Dept. of Biochemistry.
Advisor: Michael Forgac.
Committee: Andrew Bohm, Brent Cochran, and Peter Juo.
Keywords: Biochemistry, Cellular biology, and Molecular biology.read less - ID:
- 086131639
- To Cite:
- TARC Citation Guide EndNote
