Breast cancer is one of the most common causes of cancer death in females. Most patients have oestrogen-dependent disease that responds to SERMs such as tamoxifen. However, many patients either do not respond or eventually become resistant to the drug. There is a need for treatment spection markers to predict response to tamoxifen. Previously, the circadian-associated protein timeless was identified as a potential predictor of tamoxifen response, but the mechanisms underlying this association are unknown.
To investigate potential roles of timeless in oestrogen signaling, we quantified timeless expression in a panel of ER+ and ER- cell lines. We hypothesized that timeless would be higher in ER+ cells than ER-, since timeless is known to be induced by oestradiol. Our second aim was to generate stable cell lines that over-express timeless, for use in functional assays.
We quantified timeless protein and RNA expression by immunoblotting and realtime PCR in T47D, MCF7, ZR-75-1, MDA-MB231 and MDA-MB468 cells. MCF7, T47D and MDA-MB468 cells stably overexpressing timeless were generated by transfecting cells with pcDNA4Flag-Timeless and selection with Zeocin. Validation of Tim-overexpressed cells in RNA level was performed by quantitative PCR.
Timeless was expressed in both ER+ (T47D, MCF7, and ZR-75-1) and ER- (MDA-MB468, MDA-MB231) cells. No significant difference in protein and RNA expression across the cells was observed. After transfecting timeless into cells, timeless expression was increased by 3-fold, 2-fold, and 12-fold in T47D, MCF7, and MDAMB486 cells, respectively.
Timeless was expressed in both ER+ and ER- breast cancer cell lines. Therefore, although timeless is a known ER target gene, it is not dependent on ER for basal expression. Further, we successfully over expressed timeless in each cell line. These cells will aid future work to identify the effects of timeless on oestrogen signaling, cell proliferation of breast cancer cell function.