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

Oncogenesis in breast cancer is often associated with excess estrogen receptor alpha(ERalpha) activation and overexpression of its coactivators. LRP16 is both an ERalpha target gene and an ERalpha coactivator, and plays a crucial role in ERalpha activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.

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 ERalpha, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERalpha, attenuated ERalpha-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERalpha 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 ERalpha-mediated signaling and promoted cell cycle progression.

K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERalpha-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERalpha and promotes the proliferation of ERalpha-positive breast tumor cells. K18 plays an important functional role in regulating the ERalpha signaling pathway.