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

Abstract

BackgroundOncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.ResultsYeast 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 ERα, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERα, attenuated ERα-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERα 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 ERα-mediated signaling and promoted cell cycle progression.ConclusionsK18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERα-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERα and promotes the proliferation of ERα-positive breast tumor cells. K18 plays an important functional role in regulating the ERα signaling pathway.

Differentially expressed proteins in human breast cancer cells sensitive and resistant to paclitaxel

“…Relative to our results, Meng et al (73) found a lower expression of cytokeratin 18 promoting proliferation of breast cancer cells MCF-7; however, they connected this effect to the modulation of estrogen receptor-α pathway. Nevertheless, SK-BR-3 cells do not express estrogen receptor-α.…”

Biological Functions of Cytokeratin 18 in Cancer

“…Loss of CK18 expression increases the functional availability of LRP16 to ERa and promotes the proliferation of ERa-positive breast tumor cells. These findings indicate that CK18 plays an important functional role in regulating the ERa pathway (46). High-risk human papillomaviruses, such as human papillomavirus type 16 (HPV16), are the primary cause of cervical cancer.…”

Keratins in health and cancer: more than mere epithelial cell markers

“…K18 may play a regulatory role in hormonally responsive breast cancer, as it can effectively associate with and sequester the ER α target gene and ERα coactivator LRP16 in the cytoplasm, thus attenuating ERα-mediated signaling and estrogen-stimulated cell cycle progression in breast tumor cells (Meng et al , 2009). Furthermore, autophagy defects, which promote mammary tumorigenesis (Karantza-Wadsworth et al , 2007), result in K8, K17 and K19 upregulation in mouse mammary tumor cells under metabolic stress in vitro and in allograft mouse mammary tumors in vivo (Kongara et al , 2010), potentially implicating deregulation of keratin homeostasis in defective autophagy-associated breast cancer, a hypothesis worthy of further investigation.…”