Calcium influx regulators in mammary gland development and breast cancer: Roles of ORAI and STIM isoforms

Damara McAndrew (2010). Calcium influx regulators in mammary gland development and breast cancer: Roles of ORAI and STIM isoforms PhD Thesis, School of Pharmacy, The University of Queensland.

       
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Author Damara McAndrew
Thesis Title Calcium influx regulators in mammary gland development and breast cancer: Roles of ORAI and STIM isoforms
School, Centre or Institute School of Pharmacy
Institution The University of Queensland
Publication date 2010-04
Thesis type PhD Thesis
Supervisor Sarah Roberts-Thomson
Gregory Monteith
Total pages 316
Total colour pages 20
Total black and white pages 296
Subjects 11 Medical and Health Sciences
Abstract/Summary Calcium is the major mineral component of milk and is essential for neonatal development. To enrich the milk, calcium must pass from the maternal bloodstream, through mammary epithelial cells, into the alveolar lumen. While calcium extrusion from the epithelial cells is well characterized, no calcium channel or transporter has been identified as the major conduit for calcium to enter the mammary epithelial cell from the bloodstream. A major aim of this thesis was to identify a calcium channel or channels responsible for calcium influx into mammary epithelial cells during lactation. Real time reverse transcription-polymerase chain reaction was used to investigate in vivo expression of calcium channels in the murine mammary gland at the four main stages of mammary gland development. The store-operated calcium channel Orai1 was upregulated during lactation relative to its expression in the nulliparous gland. The classic ORAI1 regulator Stim1 was not similarly overexpressed during lactation, however, its isoform Stim2 was modestly upregulated. HC11 murine mammary cells were used as a model to further investigate the role of STIM2 on calcium handling during lactation. siRNA knockdown of Stim2 reduced both basal and agonist-induced peak cytosolic calcium levels, indicative of its role in calcium regulation. In addition to investigating the role of calcium channels in normal mammary development, their role in breast cancer was examined. Real time reverse transcription-polymerase chain reaction was used to identify calcium channels upregulated in human breast cancer cell lines, relative to non-tumorigenic mammary cell lines. TRPV1, TRPV6, and ORAI1 were upregulated in the breast cancer cell lines. Pharmacological modulation of ORAI1 resulted in modest changes in proliferation, but as there was no specific ORAI1 inhibitor, this effect could not be conclusively attributed to ORAI1 inhibition. siRNA was used to specifically target ORAI1 in three human breast cancer cell lines: MCF-7, MDA-MB-231, and T-47D. siRNA knockdown of ORAI1 was specific and potent, and reduced cell viability and altered calcium handing in all three cell lines. The alterations caused by ORAI1 knock down were not related to the expression of the genes CDK2 and FOS, as these did not change upon ORAI1 knockdown. Data mining was performed using the National Center for Biotechnology Information’s (NCBI’s) expressed sequence tag (EST) database, dbEST, and the Oncomine database. ORAI1 was elevated in estrogen receptor negative breast cancers and in the basal breast cancer molecular subtype, a subtype that has a poor prognosis. Other data suggested that breast cancer cells with high STIM1 and low STIM2 expression also correlated with the basal breast cancer subtype. These data indicate that ORAI and STIM proteins have a role in the physiological process of lactation as well as in the regulation of tumorigenic pathways in the breast, and particular gene expression profiles may be predictors of disease prognosis.
Keyword Calcium
breast cancer
Lactation
mammary gland
ORAI1
STIM2
Additional Notes Colour pages: 29, 41, 48, 95, 103, 105, 113, 114, 157, 207, 209, 211, 231, 236, 238, 241, 243, 247, 249

 
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Created: Fri, 25 Jun 2010, 11:30:36 EST by Ms Damara Mcandrew on behalf of Library - Information Access Service