Abstract 1707: Understanding the role of choline kinase in breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Choline kinase (Chk), the enzyme that converts choline to phosphocholine (PC) in the choline phospholipid metabolism pathway has been evaluated as a novel target since increased levels of Chk-α and PC are consistently observed in aggressive cancers including breast cancer. We previously showed that the Chk inhibitor, V-11-0711 (Vertex Pharmaceuticals (Europe) Ltd), which reduces the function of Chk-α by binding to the active site and inhibiting the catalytic activity but not affecting Chk-α protein levels, had no effect on MDA-MB-231 cell proliferation, although downregulation of Chk with siRNA (siRNA-chk) reduced proliferation. Here we have confirmed and expanded these observations in the SUM149 triple negative inflammatory breast cancer cell line. These data confirm the aberrant expression of this enzyme and provide new insights into its role in breast cancer. The cell proliferation assay (cell counting kit-8, Dojindo) detected no significant reduction of viability in SUM149 cells treated with up to 5μM V-11-0711 for 48 h. After 48 h treatment with 0.1μM and 1μM V-11-0711, Chk-α protein levels in SUM149 cells were stable. Fully relaxed 1H NMR spectroscopy of water-soluble phase of cell extracts performed on a Bruker Avance 500 spectrometer showed that levels of PC and total choline (PC + glycerophosphocholine (GPC) + free choline) decreased significantly and dose dependently after treatment with 0.1μM and 1μM V-11-0711. GPC levels decreased significantly only following treatment with 1μM V-11-0711. Treatment with 1μM V-11-0711 reduced PC to almost non-detectable level. We have previously shown that the downregulation of Chk expression significantly reduced the proliferation in breast cancer cells and tumors [1-3]. Our results here indicate that reduction of PC under these conditions does not markedly affect the proliferation of breast cancer cells if Chk-α protein levels are not reduced. To further investigate if reduced protein levels of Chk-α affect proliferation in SUM149 cells, we transfected cells with siRNA-chk. siRNA-chk reduced Chk-α protein to undetectable levels and proliferation was significantly reduced whether 0.1μM V-11-0711 was present or not. Our results demonstrate that, similar to MDA-MB-231 cells, reduction of PC has little affect on breast cancer cell proliferation as long as Chk-α protein levels are not reduced. These data are consistent with results obtained by Miyake et al., [4] on the potential role of Chk-α as a chaperone protein, and suggest that the Chk-α protein may be essential in cancer cell proliferation. The data support the development of strategies that destabilize or downregulate Chk-α protein. [1] Glunde K et al, Cancer Res, 65, (2005); [2] Mori N et al, Cancer Res, 67, (2007); [3] Krishnamachary B et al, Cancer Res, 69, (2009); [4] Miyake T et al., Oncogene (2011). This work was supported by NIH R01 CA73850 and P50 CA103175. Citation Format: Noriko Mori, Flonne Wildes, Kristine Glunde, Zaver M. Bhujwalla. Understanding the role of choline kinase in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1707. doi:10.1158/1538-7445.AM2013-1707