Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1-and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1-and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wntdriven cancer.antibody therapeutics | cancer T he Wnt/β-catenin pathway regulates diverse biological processes during development and tissue homeostasis by modulating the protein stability of β-catenin (1-3). In the absence of extracellular Wnt proteins, cytoplasmic β-catenin is associated with the β-catenin destruction complex and degraded by ubiquitinmediated proteolysis. Wnt signals are transduced by two distinct receptors, the serpentine receptor Frizzled (Frz) and the singlespan transmembrane proteins LRP5 or LRP6. Wnt proteins promote the assembly of the Frz-LRP5/6 signaling complex and induce phosphorylation of LRP5 or LRP6. Phosphorylated LRP5 or LRP6 inactivates the β-catenin degradation complex, allowing stabilized β-catenin to enter the nucleus, bind to the TCF transcription factors, and act as a transcriptional coactivator.The extracellular domain of LRP5 or LRP6 contains four YWTD-type β-propeller domains each followed by an EGF-like domain and an LDLR domain. Each propeller contains six YWTD motifs that form a six-bladed β-propeller structure (4). Biochemical studies suggest that Wnt proteins physically interact with both Frz and LRP6 and induce the formation of an Frz-
Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.The Polycomb group (PcG) genes were first identified by their roles in ensuring appropriate temporal and spatial expression of Homeotic (Hox) genes, which act as transcriptional repressors required for correct body patterning during development. Members of the PcG family also play important regulatory roles in adult organisms, including stem cell maintenance, differentiation, and malignant transformation (for a review, see references 15, 23, and 42).PcG members are generally subdivided into two groups, Polycomb repressive complex 1 (PRC1) and PRC2, and are associated with distinct multiprotein complexes (13,30). It is believed that a coordinated action between PRC1 and PRC2 complexes introduces heritable histone modifications at certain promoters, followed by the recruitment of additional transcriptional repressors to these sites. The PRC2 complex, which includes Ezh2 and EED, possesses histone H3 lysine 9 and 27 methyltransferase activity, a "silencing" histone mark (8,10,38). PRC1 components subsequently bind to methylated H3K27, inhibit SWI/SNF remodeling, and recruit histone deacetylases to silence gene expression (11,47). Biochemical histone modification by components of the PRC1 complex has also been described: reconstituted PRC1-like complexes containing Bmi1 and RING1B contain an E3 ubiquitin ligase activity capable of ubiquitinating histone H2A (7, 46).Bmi-1 and Mel-18 are highly homologous constituents of the PRC1 complex. They share 65% amino acid identity and contain a RING finger domain in the N terminus, as well as proline-rich sequences and a helix-turn-helix domain in the C-terminal tail. While they are viable and have...
HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of PI3K signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-drive tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells and it displayed single agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations and was similarly active in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope within domains 2 and 4 that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that in combination with HER2 or EGFR-targeted agents may leverage their clinical efficacy in ErbB-driven cancers.
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