The calcium (Ca2+) ion is a highly diverse intracellular signal crucial in the regulation of processes essential for cell growth and death. Intracellular Ca2+ concentrations are tightly regulated by an array of Ca2+ transporters, including channels, pumps and exchangers. Aberrant expression and activity of Ca2+ transporters is implicated in disease pathophysiology, including cancer progression. The Secretory Pathway Ca2+ ATPase (SPCA) is a Golgi-localized Ca2+ ATPase, responsible for the active transport of Ca2+ and Mn2+ ions into the Golgi apparatus, a major site of protein processing and trafficking. Two isoforms of SPCAs exist; namely SPCA1 and SPCA2. Microarray studies provide evidence for the differential expression of SPCA1 and SPCA2 in breast tumor subtypes; SPCA2 is overexpressed in luminal breast tumors while SPCA1 is overexpressed in basal- like breast cancers, a molecular subtype of breast cancer that infers a poor prognosis. The work in this thesis describes the role of SPCA1 in a basal breast cancer cell line, MDA-MB-231.
Accumulating evidence suggests that anomalous alternative splicing of some proteins is associated with epithelial-to-mesenchymal transition (EMT), and that specific patterns of alternative splicing are characteristic of particular subtypes of cancer. Although SPCA1 has four alternative splice variants, there is a paucity of data regarding their expression in the context of EMT and in relation to molecular subtypes of breast cancer. SPCA1 splice variant expression was assessed in an EGF-induced model of EMT, and also in a panel of breast cancer cell lines, classified into the basal and luminal subtypes of breast cancer. The results did not reveal any specific correlation between altered splicing patterns of SPCA1 in EMT, or in basal and luminal breast cancer cell lines.
Recent findings suggest SPCA1 supplies Ca2+ and Mn2+ ions essential for the activity of Golgi-resident enzymes, and have demonstrated that the silencing of SPCA1 alters the post-translational modification of IGF-1R, a receptor strongly implicated in tumor growth and invasiveness. The work in this thesis focused on identifying other proteins sensitive to SPCA1 inhibition. Beginning with an individualized target approach, this work used immunoblotting to study the expression and post-translational modification of five proteins that undergo extensive post-translational modification, and are important in cancer pathways, including MUC-1, MMP-9 and CD147. No change in the post-translational modification or expression of these proteins was detected with SPCA1 silencing in MDA- MB-231 cells.
These results prompted a switch to a high-throughput approach, using a technique in proteomics known as 2 dimensional difference in gel electrophoresis (2D-DIGE). Validation of results from a 2D-DIGE analysis identified a significant downregulation of heat shock protein 60 (HSP60) protein expression with SPCA1 silencing, a molecular chaperone associated with an array of cellular signaling pathways. Work in this thesis also presents results further investigating the mechanisms behind SPCA1 silencing-mediated downregulation of HSP60, and explores the functional consequences of SPCA1 silencing on staurosporine-induced and heat shock-induced cell death pathways. SPCA1 silencing resulted in the transcriptional downregulation of HSP60, possibly via an NFκβ-mediated pathway. Silencing of SPCA1 in MDA-MB-231 cells also altered sensitivity to staurosporine-induced cell death. 2D-DIGE also identified β2C-tubulin and transketolase as protein targets sensitive to SPCA1 silencing in MDA-MB-231 cells.
The results presented in this thesis collectively demonstrate that SPCA1 is not a global regulator of post-translational modification pathways in breast cancers, but rather, controls specific pathways of protein modification and expression. Using 2D-DIGE technology, these results demonstrate for the first time in human breast cancer cells that the silencing of SPCA1 leads to the transcriptional and translational downregulation of HSP60, a protein strongly implicated in cell death and apoptotic pathways. Given the upregulation of SPCA1 in basal breast tumors, this work may lead to a better understanding of the role of SPCA1 in pathways contributing to cancer development.