P4-01-17: TIMP-1 Over-Expression Confers Resistance of MCF-7 Breast Cancer Cells to Fulvestrant.
Authors: CA Bjerre, L Vinther, K Belling, Rasmussen A-S Schrohl, J Li, X Lin, Z Han, J Wang, L Bolund, V Jensen, BS Nielsen, R Soekilde, R Gupta, U Lademann, N Brünner, J Stenvang
Journal: Cancer Research (2011)

Abstract Background Endocrine resistance represents a major challenge in the management of estrogen receptor (ER) positive breast cancer. Currently no predictive biomarkers for endocrine resistance in ERpositive breast cancer patients are in clinical use. In a clinical study, patients with metastatic breast cancer and high levels of serum Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) had less benefit from endocrine therapy than patients with a lower level of serum TIMP-1 [1]. Therefore, we evaluated the association between TIMP-1 and response to endocrine therapy using an in vitro approach. We have previously presented initial results on TIMP-1 and response to endocrine therapy [2]. Materials and Methods: MCF-7 cells were stably transfected with pcDNA3.1(Hyg)-TIMP-1 plasmid, and a panel of 11 subclones with different expression levels of TIMP-1 was generated. TIMP-1 expression levels were confirmed using enzyme-linked immunosorbent assay (ELISA). Four subclones with high or low TIMP-1 expression were analyzed for the growth response to estrogen, 4-hydroxytamoxifen and fulvestrant. These four subclones were analyzed for protein expression by western blotting. All subclones were analyzed by whole human genome oligo microarrays 4×44K for determination of gene expression levels. Data were analyzed using the limma R/Bioconductor package. Paired-end Solexa sequencing was applied to selected subclones with high and low TIMP-1 levels to identify transcriptomic changes. Results: High expression of TIMP-1 was associated with resistance to fulvestrant, whereas growth response to either estrogen or 4-hydroxytamoxifen was independent of TIMP-1 expression levels. High expression of TIMP-1 protein and mRNA was associated with undetectable levels of progesterone receptor (PgR) protein and mRNA whereas ER protein and mRNA levels were unaffected by TIMP-1. To characterize the potential role of TIMP-1 in estrogen signaling we analyzed the expression of reported estrogen-responsive genes and no general change was observed. We identified genes that correlated positively or negatively to TIMP-1 expression. Among the identified genes was PgR, which is a direct target for ER. Conclusion: Our data suggest that a high expression of TIMP-1 in vitro is associated with resistance to fulvestrant but not to 4-hydroxytamoxifen. Estrogen-regulated genes are not generally affected by changes in TIMP-1 expression levels and therefore TIMP-1 appears to affect endocrine resistance through other mechanisms than globally regulating ER signaling. However, high expression of TIMP-1 is associated with loss of PgR and this may be related to the resistance towards fulvestrant. References: [1] Lipton, A et al: Serum TIMP-1 and Response to the Aromatase Inhibitor Letrozole Versus Tamoxifen in Metastatic Breast Cancer. J Clin Oncol; 2008; 26;(16); 2653–8 [2] Effect of TIMP-1 Overexpression on Endocrine Sensitivity of MCF-7 ER-positive Human Breast Cancer Cells In Vitro. Cancer Res; 2009; 69(24 suppl); abstract nr 2029 Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-17.