Intracellular calcium signaling in a model of breast cancer metastasis

Felicity Davis (2012). Intracellular calcium signaling in a model of breast cancer metastasis PhD Thesis, School of Pharmacy, The University of Queensland.

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Author Felicity Davis
Thesis Title Intracellular calcium signaling in a model of breast cancer metastasis
School, Centre or Institute School of Pharmacy
Institution The University of Queensland
Publication date 2012-01
Thesis type PhD Thesis
Total pages 329
Total colour pages 16
Total black and white pages 313
Language eng
Subjects 111207 Molecular Targets
060111 Signal Transduction
111201 Cancer Cell Biology
Abstract/Summary Metastasis is the major cause of mortality in women with breast cancer. Epithelial-mesenchymal transition (EMT) is a pathway implicated in metastasis, which describes the transition from an epithelial to a mesenchymal (invasive) cell phenotype. This involves the down-regulation of junctional proteins including E-cadherin; re-organization of the cytoskeleton and induction of vimentin; and changes in cell shape to a more spindle-like morphology. This phenotypic switch facilitates cancer cell migration and invasion. This thesis explores roles for Ca2+ signaling during EMT in breast cancer cells. Ca2+ is important for many processes, ranging from neurotransmission to cardiac contractility. While many studies have now established roles for Ca2+ in cancer cell proliferation, apoptosis and differentiation, a role for Ca2+ in EMT has not been assessed. To explore changes in Ca2+ homeostasis during EMT, an inducible model of EMT was established in MDA-MB-468 human breast cancer cells. These cells over-express the epidermal growth factor (EGF) receptor and have previously been reported to undergo EMT in response to EGF stimulation. EMT in MDA-MB-468 cells was confirmed by assessing changes in cell morphology as well as molecular markers of EMT, including E-cadherin, snail, twist, and vimentin. The first part of this thesis examines the question—is the conversion to a more mesenchymal and invasive phenotype through EMT associated with a remodeling of Ca2+ transport protiens and/or altered Ca2+ homeostasis? To address this question, changes in Ca2+ signaling mediated by the activation of specific cell surface receptors were assessed in MDA-MB-468 breast cancer cells induced to undergo EMT. A 10-fold shift in the potency of ATP was observed, in addition to alterations in the nature of the ATP-induced Ca2+ transient. No change in the sensitivity to protease-activated receptor (PAR2)-mediated Ca2+ signaling was observed. To determine whether changes in Ca2+ signaling are preceded by alterations in the transcriptional profile of purinergic ATP receptors, the expression of a panel of 15 ionotropic (P2X) and metabotropic (P2Y) purinergic receptors were analyzed. Significant alterations in the transcription of ATP-activated purinergic receptors were observed as a consequence of EGF-induced EMT in MDA-MB-468 cells, the most pronounced of which was an up-regulation of P2X5. P2X5 levels were also elevated in the basal-like subtype of human breast cancers, where the mesenchymal phenotype is over-represented. To further explore possible changes in Ca2+ homeostasis associated with EMT, alterations in store-operated and agonist-stimulated Ca2+ entry pathways were assessed in MDA-MB-468 breast cancer cells induced to undergo EMT with EGF. Surprisingly, a high degree of non-stimulated Ca2+ influx (Ca2+ influx in the absence of agents to deplete endoplasmic reticulum (ER) Ca2+ reserves) was observed in MDA-MB-468 breast cancer cells. This non-stimulated Ca2+ influx was significantly attenuated as a consequence of EGF-induced EMT. Reduced agonist-stimulated and store-operated Ca2+ entry were also a feature of the more mesenchymal phenotype. Silencing of the canonical-type transient receptor potential (TRPC1) channel reduced non-stimulated Ca2+ influx in MDA-MB-468 breast cancer cells. However, silencing of the store-operated Ca2+ entry channel ORAI1 largely abolished non-stimulated Ca2+ influx in these cells, in addition to producing large reductions in agonist-stimulated and store-operated Ca2+ entry. Although not a regulator of store-operated Ca2+ entry in these cells, silencing of TRPC1 (but not ORAI1) greatly altered ER Ca2+ release kinetics. These results define distinct but intersecting roles for TRPC1 and ORAI1 in MDA-MB-468 breast cancer cells and a remodeling of Ca2+ influx with EGF-induced EMT. Finally, alterations in the expression of intracellular Ca2+ store channels, pumps and exchangers with EGF-induced EMT were assessed. No changes in the level of Golgi, endolysosomal or mitochondrial Ca2+ channels, pumps or exchangers were detected. However, EGF-induced EMT in MDA-MB-468 breast cancer cells was associated with significant alterations in the transcription of ER Ca2+ channels/pumps, including sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps, inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RYRs). The most pronounced change observed was a > 46-fold increase in the transcription of RYR2. The second part of this thesis addresses the question—does Ca2+ signaling regulate the induction of EMT by EGF in breast cancer cells? Preliminary studies found that common techniques used to induce EMT, including scratch-wounding and EGF treatment, also elicited an intracellular Ca2+ response. Intracellular Ca2+ chelation with BAPTA-AM inhibited EGF-induced vimentin induction in MDA-MB-468 cells. In addition, STAT3, an important signal-transducer that is required for EGF-induced EMT in MDA-MB-468 cells, was shown to be highly Ca2+-dependent. To characterize Ca2+ channels that may regulate Ca2+-dependent EMT, a targeted siRNA-based screen was performed. These results identified TRPM7 Ca2+ channels as regulators of EMT induction, possibly via control of STAT3 activity. In conclusion, this work describes a remodeling of Ca2+ transport proteins and altered Ca2+ homeostasis associated with breast cancer EMT, and establishes a central role for Ca2+ signaling in EMT induction. Given the role for EMT in cancer metastasis and the attractiveness of Ca2+ channels/pumps as drug targets, this work may aid in the identification of therapeutic targets for preventing cancer metastasis.
Keyword Breast cancer
Epithelial-Mesenchymal Transition
calcium signaling
Signal transduction
Epidermal Growth Factor
Gene Expression
Calcium imaging
Additional Notes 55, 99, 103, 105, 124, 143, 148, 179, 198, 202, 223, 236, 239, 256, 262, 273

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Created: Fri, 13 Apr 2012, 12:43:40 EST by Felicity Davis on behalf of Library - Information Access Service