Abstract 3301: Inhibition of the secretory pathway calcium ATPase 1 (SPCA1) in MDA-MB-231 breast cancer cells and the effect on protein expression

Abstract

SPCA1 is a Ca2+ pump located on the membrane of the Golgi apparatus and is involved in controlling calcium levels within the Golgi. We have previously shown that SPCA1 is upregulated in some basal-like breast cancers. Given the role of the Golgi in protein trafficking and processing, we hypothesised that SPCA1 may be involved in regulating the expression of proteins important in cancer. To address this hypothesis we used 2D-DIGE to identify proteins that are altered when SPCA1 is silenced in MDA-MB-231 basal cancer cells. MDA-MB-231 cells were seeded into 6-well plates (75,000 cells/well) and treated with SPCA1 or non-targeting siRNA. Protein was isolated 72 h post siRNA treatment, and SPCA1 silencing was confirmed by immunoblotting. 2D-DIGE and MS/MS were used to identify proteins with differential expression with SPCA1 silencing. Heat shock protein 60 (HSP60) was identified as a protein potentially sensitive to SPCA1 silencing using 2D-DIGE. Immunoblotting and real time RT-PCR showed that SPCA1 silencing reduced HSP60 protein and mRNA expression by 81±2% and 60±7% (n=3, P<0.05), respectively. Given the role of HSP60 in cell death pathways, we also assessed the consequences of SPCA1 silencing on heat shock and staurosporine-induced cell death. SPCA1 silencing had no effect on heat shock-induced cell death but reduced the sensitivity of MDA-MB-231 breast cancer cells to staurosporine. Although further studies are required to fully characterize the functional consequences of HSP60 downregulation upon SPCA1 silencing, this work suggests that SPCA1 is a complex regulator of proteins important in cancer pathways. Citation Format: Jane M. Lee, Diana G. Ross, Gregory R. Monteith, Sarah J. Roberts-Thomson. Inhibition of the secretory pathway calcium ATPase 1 (SPCA1) in MDA-MB-231 breast cancer cells and the effect on protein expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3301. doi:10.1158/1538-7445.AM2014-3301