Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm

Background: Oncogenesis in breast cancer is often associated with excess estrogen receptor alpha(ER alpha) activation and overexpression of its coactivators. LRP16 is both an ER alpha target gene and an ER alpha coactivator, and plays a crucial role in ER alpha activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear. Results: Yeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP) identified the cytoplasmic intermediate filament protein keratin 18 (K18) as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ER alpha, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ER alpha, attenuated ER alpha-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ER alpha recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ER alpha-mediated signaling and promoted cell cycle progression. Conclusions: K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ER alpha-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ER alpha and promotes the proliferation of ER alpha-positive breast tumor cells. K18 plays an important functional role in regulating the ER alpha signaling pathway.