Abstract
BackgroundLong-term estrogen deprivation models are widely employed in an in vitro setting to recapitulate the hormonal milieu of breast cancer patients treated with endocrine therapy. Despite the wealth information we have garnered from these models thus far, a comprehensive time-course analysis of the estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER-2/neu) receptors on the gene and protein level, coupled with expression array data is currently lacking. We aimed to address this knowledge gap in order to enhance our understanding of endocrine therapy resistance in breast cancer patients.MethodsER positive MCF7 and BT474 breast cancer cells were grown in estrogen depleted medium for 10 months with the ER negative MDA-MB-231 cell line employed as control. ER, PR and HER-2/neu expression were analysed at defined short and long-term time points by immunocytochemistry (ICC), and quantitative real-time RT-PCR (qRT-PCR). Microarray analysis was performed on representative samples.ResultsMCF7 cells cultured in estrogen depleted medium displayed decreasing expression of ER up to 8 weeks, which was then re-expressed at 10 months. PR was also down-regulated at early time points and remained so for the duration of the study. BT474 cells generally displayed no changes in ER during the first 8 weeks of deprivation, however its expression was significantly decreased at 10 months. PR expression was also down-regulated early in BT474 samples and was absent at later time points. Finally, microarray data revealed that genes and cell processes down-regulated in both cell lines at 6 weeks overlapped with those down-regulated in aromatase inhibitor treated breast cancer patients.ConclusionsOur data demonstrate that expression of ER, PR, and cell metabolic/proliferative processes are unstable in response to long-term estrogen deprivation in breast cancer cell lines. These results mirror recent clinical findings and again emphasize the utility of LTED models in translational research.
Highlights
Long-term estrogen deprivation models are widely employed in an in vitro setting to recapitulate the hormonal milieu of breast cancer patients treated with endocrine therapy
Control and Long-term estrogen deprived (LTED) cells were routinely maintained in phenol red containing MEM or DMEM supplemented with 10% fetal bovine serum (FBS) or phenol red-free MEM or DMEM supplemented with 10% dextran-coated charcoal-stripped FBS (DCC-FBS) to remove substantial amounts of estrogen, respectively
Re-expression of ER in an estrogen deprived environment occurs in the absence of PR in MCF7 cells The breast cancer cell lines MCF7 and BT474 were cultured without estrogen for up to 10 months and examined by immunocytochemistry and quantitative real-time RTPCR (qRT-PCR) for changes in expression of ER, PR and HER-2/neu at the time points shown in our experimental overview (Figure 1)
Summary
Long-term estrogen deprivation models are widely employed in an in vitro setting to recapitulate the hormonal milieu of breast cancer patients treated with endocrine therapy. Estrogens are steroid hormones that play important roles in the growth and development of Approximately two-thirds of all breast cancer tumours are ER-positive [4,5,6] and more than 50% of these are PR-positive [7]. Both receptors are useful in predicting response to endocrine therapy [5,7,8,9] and in general ER-negative tumours are associated with early recurrence and poor patient survival relative to those that are ER-positive [5,8,9]. Complicating matters, we and others have shown in mostly retrospective studies, that expression of ER and PR are unstable during tumour progression from a primary lesion to its corresponding metastasis [10,11,12,13]
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