LDL receptor-related protein 6 (LRP6) in Wnt signaling and breast cancer tumorigenesis.

摘要

Breast cancer is a diverse malignancy; understanding the molecular mechanisms driving tumor progression will facilitate development of targeted therapies. For example, tamoxifen, an anti-estrogen agent, used for treating estrogen receptor (ER)-positive breast tumors, and trastuzumab, a monoclonal antibody targeting HER2-overexpressing tumors, have benefitted specific subsets of breast cancer patients. However, the enormous variation in breast cancer initiation and growth complicate biological approaches to this malignancy and challenge the effectiveness of current therapies. Elucidation of novel, targetable signaling pathways in breast cancers would provide means of addressing current deficiencies in the treatment of the disease.;The Wnt signaling pathway is involved in various differentiation events during embryonic development and can lead to tumor formation when aberrantly activated. Aberrant Wnt activation is found in 40-60% of breast cancers. However, because mutations in the classical intracellular components of the Wnt signaling pathway are rare, the underlying cause of this aberrant activation remains elusive and may involve dysregulation of upstream elements. In this thesis, we found that overexpression of LRP6, a Wnt signaling co-receptor, plays a crucial role in the pathogenesis of a subtype of human breast tumor. LRP6 was increased more frequently in ER or HER2-negative or triple-negative breast tumors that are either harder to treat or more likely to recur. We further demonstrated that suppression of LRP6 expression and function significantly inhibits Wnt signaling and breast tumor growth in vitro and in vivo.;Mesd (mesoderm development) was previously identified as a specialized chaperone of LRP5/6. Using NMR structural and molecular tools, we first showed that the core chaperone domain of Mesd is required for the proper folding of LRP5/6, while its C-terminal escort domain ensures a safe trafficking of LRP5/6 from the ER to Golgi by preventing premature ligand binding. With the goal of developing a novel strategy of inhibiting Wnt signaling at the cell surface, we further demonstrated that Mesd could serve as an LRP5/6 inhibitor to reduce tumor burden in Wnt-driven breast cancer without causing undesirable side effects. Together, our studies highlight LRP6 as a potential therapeutic target in breast cancer, and introduce Mesd as a promising antitumor agent for treating breast cancer subtypes with Wnt activation at the cell surface.;Trafficking of receptors to the cell surface, ligand/antagonists modulations, and endocytosis of cell surface receptors are important regulatory events of signal transduction pathways. LRP6 consists of four beta-propeller domains flanked by EGF repeats (BP) at its extracellular region. In this thesis, we extensively analyzed the folding and ligand-binding properties of LRP6 BP. The cooperative folding and maturation were observed between BP1 & BP2 and BP3 & BP4. BP12 and BP34 serve as functional units displaying different ligand binding preferences. Another aim of this thesis is to identify the endocytosis motifs of LRP6 at its cytoplasmic tail and investigate the relationship between LRP6 endocytic trafficking and Wnt signaling activation. Using flow cytometry, immunofluorecent staining, and endocytosis assay, we showed that LRP6 mutant with defective tyrosine-based signals (Y mutant) in its cytoplasmic tail displayed increased cell surface distribution. We further found that Wnt3a ligands and mutation of tyrosine-based signals in LRP6 tail redistribute this receptor to lipid rafts, which are membrane microdomains that consist of a dynamic assembly of cholesterol and glycosphingolipids. LRP6 Y mutant also exhibited higher signaling in response to Wnt3a ligand stimulation compared to wild-type, and this activation was blocked when cells were treated with Nystatin, a lipid raft-disrupting agent that inhibits caveolae-mediated endocytosis. Our results suggest that tyrosine-based signals serve as an endocytosis motif in LRP6 tail and play an important role in the determination of LRP6 internalization route and Wnt signaling activation.;Taken together, our studies provide a comprehensive appreciation for the dynamic regulation and pathological roles of LRP6 in Wnt signaling and tumorigenesis.