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

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 celldeath, inhibition of PMCA-mediated Ca2+ efflux has been proposed as a therapeutic strategy to killbreast 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-231breast cancer cell line. These studies were performed in human MDA-MB-231 breast cancer cells, awidely 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) andplasma membrane integrity (propidium iodide fluorescence) were assessed in MDA-MB-231 cellsusing ionomycin (Ca2+ ionophore), and ABT-263 (B-cell lymphoma-2 (Bcl-2) inhibitor) to activatecaspase-independent and caspase-dependent cell death pathways, respectively. The consequences ofPMCA1 or PMCA4 silencing on cell death, along with their effects on [Ca2+]CYT and nuclear factorkappa-B (NFkB) nuclear translocation were evaluated. Initial studies demonstrated that in the absenceof 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 silencingpromoted ABT-263 (caspase-dependent)-induced cell death, independent of global [Ca2+]CYTsignalling. Assessment of NFkB nuclear translocation showed that PMCA4 knockdown, but notPMCA1 silencing, attenuated transcription factor activity. The ability for the NFkB inhibitor IMD-0354 to phenocopy the effect of PMCA4 siRNA on the promotion of ABT-263 cell death was alsodemonstrated. 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 deathresponses in the MDA-MB-231 breast cancer cell line.Additional experiments examined in more detail, PMCA1-mediated regulation of global [Ca2+]CYTgenerated by different GPCR activators and the potential for SERCA (sarco/endoplasmic reticulumCa2+ ATPase) activity to compensate for PMCA1 knockdown. These experiments showed thatPMCA1 siRNA has distinct effects on ATP, compared with trypsin-induced Ca2+ responses. As apotential mechanism to explain why trypsin-mediated Ca2+ responses, compared with those of ATPare not altered by PMCA1 silencing, the potential contribution of SERCA activity was assessed usingthe SERCA inhibitor cyclopiazonic acid (CPA). PMCA4 silencing or CPA alone did not alter theshape 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 identityof the GPCR activated.PMCA2 overexpression in some breast cancers, and a report that recombinant PMCA2overexpression protects T47D breast cancer cells from Ca2+-induced (ionomycin) cell death suggeststhat this particular PMCA isoform may be a therapeutic target for the treatment of some breastcancers. To examine PMCA2 inhibition as a therapeutic strategy, the consequences of PMCA2siRNA on global [Ca2+]CYT signals and cell death responses were characterised in MDA-MB-231cells. [Ca2+]CYT signals generated with various agents demonstrated that endogenously expressedPMCA2 does not play a major role in global Ca2+ signalling. PMCA2 silencing alone produced nochange in cell viability. The effect of PMCA2 siRNA on cell death was then measured in response toionomycin 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 inhibitionmay 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 themitochondrial Ca2+ uniporter (MCU), which facilitates mitochondrial Ca2+ uptake. Molecularidentification of MCU made it possible to extend these thesis studies and examine MCU distributionin clinical breast cancers. MCU levels were elevated in the oestrogen receptor-negative and basallikesubtypes of breast cancer. The significance of this elevation was addressed by down-regulatingMCU 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 effecton 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 MCUknockdown. Inhibitors of PMCA1 or MCU may sensitise some breast tumors to cancer therapies thatactivate caspase-independent cell death. Alternatively, specific inhibitors of PMCA4 or PMCA2 mayhave therapeutic potential in sensitising some aggressive breast tumors, to cancer therapies that workthrough caspase-dependent apoptosis via the Bcl-2 cell survival pathway.