Regulation of MDA-MB-231 breast cancer cell death by plasma membrane calcium ATPase isoforms and the mitochondrial calcium uniporter

Curry, Merril Carmel (2017). Regulation of MDA-MB-231 breast cancer cell death by plasma membrane calcium ATPase isoforms and the mitochondrial calcium uniporter PhD Thesis, School of Pharmacy, The University of Queensland. doi:10.14264/uql.2017.418

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Author Curry, Merril Carmel
Thesis Title Regulation of MDA-MB-231 breast cancer cell death by plasma membrane calcium ATPase isoforms and the mitochondrial calcium uniporter
School, Centre or Institute School of Pharmacy
Institution The University of Queensland
DOI 10.14264/uql.2017.418
Publication date 2017-03-16
Thesis type PhD Thesis
Supervisor Gregory Monteith
Peter Cabot
Sarah Roberts-Thomson
Ben Ross
Irina Vetter
Total pages 201
Language eng
Subjects 0601 Biochemistry and Cell Biology
1112 Oncology and Carcinogenesis
1115 Pharmacology and Pharmaceutical Sciences
Formatted abstract
Plasma membrane calcium (Ca2+) ATPase isoforms (PMCA1-4) extrude cytoplasmic free calcium
([Ca2+]CYT) and maintain low intracellular Ca2+ levels. Because [Ca2+]CYT increases can trigger cell
death, inhibition of PMCA-mediated Ca2+ efflux has been proposed as a therapeutic strategy to kill
breast cancer cells. The principal aim of this thesis was to characterise, by silencing techniques,
PMCA isoforms and their effects on [Ca2+]CYT signals and cell death responses in the MDA-MB-231
breast cancer cell line. These studies were performed in human MDA-MB-231 breast cancer cells, a
widely studied model of basal-like breast cancer subtypes, that express the PMCA isoforms PMCA1,
PMCA4 and PMCA2.

For the measurement of cell death, changes in nuclear morphology (Hoechst 33342 fluorescence) and
plasma membrane integrity (propidium iodide fluorescence) were assessed in MDA-MB-231 cells
using ionomycin (Ca2+ ionophore), and ABT-263 (B-cell lymphoma-2 (Bcl-2) inhibitor) to activate
caspase-independent and caspase-dependent cell death pathways, respectively. The consequences of
PMCA1 or PMCA4 silencing on cell death, along with their effects on [Ca2+]CYT and nuclear factor
kappa-B (NFκB) nuclear translocation were evaluated. Initial studies demonstrated that in the absence
of a stimulus neither PMCA1 nor PMCA4 silencing altered cell viability. PMCA1 knockdown,
however, potentiated ionomycin (caspase-independent)-induced cell death and augmented global
[Ca2+]CYT signals generated with various Ca2+ mobilising agents. On the contrary, PMCA4 silencing
promoted ABT-263 (caspase-dependent)-induced cell death, independent of global [Ca2+]CYT
signalling. Assessment of NFκB nuclear translocation showed that PMCA4 knockdown, but not
PMCA1 silencing, attenuated transcription factor activity. The ability for the NFκB inhibitor IMD-
0354 to phenocopy the effect of PMCA4 siRNA on the promotion of ABT-263 cell death was also
demonstrated. This data support diversity amongst PMCA isoforms expressed in the same cell,
identifying differential roles for PMCA1 and PMCA4 in the regulation of Ca2+ signals and cell death
responses in the MDA-MB-231 breast cancer cell line.

Additional experiments examined in more detail, PMCA1-mediated regulation of global [Ca2+]CYT
generated by different GPCR activators and the potential for SERCA (sarco/endoplasmic reticulum
Ca2+ ATPase) activity to compensate for PMCA1 knockdown. These experiments showed that
PMCA1 siRNA has distinct effects on ATP, compared with trypsin-induced Ca2+ responses. As a
potential mechanism to explain why trypsin-mediated Ca2+ responses, compared with those of ATP
are not altered by PMCA1 silencing, the potential contribution of SERCA activity was assessed using
the SERCA inhibitor cyclopiazonic acid (CPA). PMCA4 silencing or CPA alone did not alter the
shape of trypsin or ATP Ca2+ responses. In combination, PMCA1 siRNA and CPA delayed trypsin-
mediated Ca2+ clearance. These results indicate that the calcium pumps, PMCA1 and SERCA,
expressed in the same cell, can differentially contribute to Ca2+ clearance depending on the identity
of the GPCR activated.

PMCA2 overexpression in some breast cancers, and a report that recombinant PMCA2
overexpression protects T47D breast cancer cells from Ca2+-induced (ionomycin) cell death suggests
that this particular PMCA isoform may be a therapeutic target for the treatment of some breast
cancers. To examine PMCA2 inhibition as a therapeutic strategy, the consequences of PMCA2
siRNA on global [Ca2+]CYT signals and cell death responses were characterised in MDA-MB-231
cells. [Ca2+]CYT signals generated with various agents demonstrated that endogenously expressed
PMCA2 does not play a major role in global Ca2+ signalling. PMCA2 silencing alone produced no
change in cell viability. The effect of PMCA2 siRNA on cell death was then measured in response to
ionomycin or ABT-263. PMCA2 silencing did not significantly alter ionomycin-induced cell death,
but potentiated ABT-263-induced apoptosis. These studies provide evidence that PMCA2 inhibition
may sensitise some breast cancer subtypes to apoptosis initiated through Bcl-2 inhibition.
Another calcium transporter that may be critical in regulating MDA-MB-231 cell death is the
mitochondrial Ca2+ uniporter (MCU), which facilitates mitochondrial Ca2+ uptake. Molecular
identification of MCU made it possible to extend these thesis studies and examine MCU distribution
in clinical breast cancers. MCU levels were elevated in the oestrogen receptor-negative and basallike
subtypes of breast cancer. The significance of this elevation was addressed by down-regulating
MCU expression in MDA-MB-231 cells and assessing the functional consequence on proliferation,
cell death and [Ca2+]CYT signals. MCU siRNA produced no change in proliferation and had no effect
on cell viability. Cell death initiated with ABT-263 was not altered by MCU silencing. MCU downregulation,
however, acted as a sensitiser of cell death triggered by ionomycin. Assessment of
[Ca2+]CYT signals indicates that MCU siRNA-mediated regulation of ionomycin-mediated cell death,
in contrast with the PMCA1 silencing studies, occurs without effects on global [Ca2+]CYT signals.

In summary, breast cancer death responses are modulated by isoform-specific PMCA or MCU
knockdown. Inhibitors of PMCA1 or MCU may sensitise some breast tumors to cancer therapies that
activate caspase-independent cell death. Alternatively, specific inhibitors of PMCA4 or PMCA2 may
have therapeutic potential in sensitising some aggressive breast tumors, to cancer therapies that work
through caspase-dependent apoptosis via the Bcl-2 cell survival pathway.
Keyword Calcium
Plasma membrane calcium ATPase
Mitochondrial calcium uniporter
Breast cancer
Cell death
G protein-coupled receptors (GPCR)
Caspase-dependent cell death
Signal transduction

Document type: Thesis
Collections: UQ Theses (RHD) - Official
UQ Theses (RHD) - Open Access
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Created: Wed, 08 Mar 2017, 07:47:36 EST by Merril Carmel Curry on behalf of University of Queensland Graduate School