Nonphosphorylatable PEA15 mutant inhibits epithelial-mesenchymal transition in triple-negative breast cancer partly through the regulation of IL-8 expression.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks targeted therapies. Patients with TNBC have a very poor prognosis because the disease often metastasizes. New treatment approaches addressing drivers of metastasis and tumor growth are crucial to improving patient outcomes. Developing targeted gene therapy is thus a high priority for TNBC patients. PEA15 (phosphoprotein enriched in astrocytes, 15 kDa) is known to bind to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the function and impact of phosphorylation status of PEA15 in the regulation of TNBC metastasis and in epithelial-to-mesenchymal transition (EMT) are not well understood. METHODS: We established stable cell lines overexpressing nonphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) mutants. To dissect specific cellular mechanisms regulated by PEA15 phosphorylation status, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to determine the effects of PEA15 phosphorylation on tumor growth and metastasis. RESULTS: We found that the nonphosphorylatable mutant PEA15-AA prevented formation of mammospheres and expression of EMT markers in vitro and decreased tumor growth and lung metastasis in in vivo experiments when compared to control, PEA15-WT and phosphomimetic PEA15-DD. However, phosphomimetic mutant PEA15-DD promoted migration, mesenchymal marker expression, tumorigenesis, and lung metastasis in the mouse model. PEA15-AA-mediated inhibition of breast cancer cell migratory capacity and tumorigenesis was the partial result of decreased expression of interleukin-8 (IL-8). Further, we identified that expression of IL-8 was possibly mediated through one of the ERK downstream molecules, Ets-1. CONCLUSIONS: Our results show that PEA15 phosphorylation status serves as an important regulator for PEA15's dual role as an oncogene or tumor suppressor and support the potential of PEA15-AA as a therapeutic strategy for treatment of TNBC.

Targeted delivery of a human Bcl-2-specific protein binder effectively induces apoptosis of cancer cells.

Bcl-2 family proteins are critical switches to control cell death and survival, and Bcl-2 is a key regulator in pro-survival signaling, causing various diseases including cancers. Bcl-2 has drawn a considerable attention as a potential target for developing a pro-apoptotic agent for cancers. We here present the development of a specific protein binder against human Bcl-2 and its cytosolic delivery to effectively induce apoptosis of cancer cells. The protein binder composed of leucine-rich repeat modules was selected for human Bcl-2, and its binding affinity was increased up to 60 nM through a modular evolution-based approach. The protein binder was efficiently delivered into cancer cells by an intracellular delivery system using a translocation domain from a bacterial exotoxin, resulting in a strong suppression of anti-apoptotic signaling in cancer cells. Our results demonstrate that the human Bcl-2-specific protein binder can act as a potent therapeutic agent for cancers.

A regulatory SH2 domain-targeting protein binder effectively inhibits the activity of Bruton's tyrosine kinase and its drug-resistant variants.

Human Bruton's tyrosine kinase (hBtk) plays a key role in growth and metabolism of B cells, but its dysfunctions cause various B-cell malignancies. Inhibitors targeting the ATP-binding pocket of hBtk have been developed, but they have several drawbacks such as adverse side effects and occurrence of drug-resistant mutations. Here, we present a protein binder which specifically binds to an allosteric regulatory SH2 domain of hBtk. The protein binder effectively inhibited the hBtk activity, indicating a critical role of the SH2 domain in allosteric regulation of the hBtk activity. Cytosolic delivery of the protein binder led to a significant inhibition on the BCR-mediated signaling and viability of B lymphoma cells. The utility of our approach was demonstrated by effective inhibition of drug-resistant hBtk variants by the protein binder. Based on the computationally predicted binding mode, the protein binder is likely to inhibit the hBtk activity by disrupting the interaction between the SH2 domain and kinase domain. The present approach can be used for developing therapeutic agents with improved efficacy for B-cell lymphoma.

Effective inhibition of C3a-mediated pro-inflammatory response by a human C3a-specific protein binder.

Complement component 3a (C3a) plays a crucial role in the immune response and host defense, but it is also involved in pro-inflammatory responses, causing many inflammatory disorders. Blockade of C3a has been regarded as a potent therapeutic strategy for inflammatory diseases. Here, we present the development of a human C3a (hC3a)-specific protein binder, which effectively inhibits pro-inflammatory responses. The protein binder, which is composed of leucine-rich repeat modules, was selected against hC3a through phage display, and its binding affinity was matured up to 600 pM by further expanding the binding interface in a module-by-module manner. The developed protein binder was shown to have more than 10-fold higher specificity to hC3a compared with human C5a, exhibiting a remarkable suppression effect on pro-inflammatory response in monocyte, by blocking the interaction between hC3a and its receptor. The hC3a-specific protein binder is likely to have a therapeutic potential for C3a-mediated inflammatory diseases.

Engineering and cytosolic delivery of a native regulatory protein and its variants for modulation of ERK2 signaling pathway.

The modulation of a cell signaling process using a molecular binder followed by an analysis of the cellular response is crucial for understanding its role in the cellular function and developing pharmaceuticals. Herein, we present the modulation of the ERK2-mediated signaling pathway through the cytosolic delivery of a native regulatory protein for ERK2, that is, PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa), and its engineered variants using a bacterial toxin-based delivery system. Based on biochemical and structural analyses, PEA-15 variants with different phosphorylation sites and a high affinity for ERK2 were designed. Semi-rational approach led to about an 830-fold increase in the binding affinity of PEA-15, resulting in more effective modulation of the ERK2-mediated signaling. Our approach enabled an understanding of the cellular function of the ERK2-mediated signaling process and the effect of PEA-15 phosphorylation on its action as an ERK2 blocker. We demonstrated the utility and potential of our approach by showing an efficient cytosolic delivery of these PEA-15 variants and the effective suppression of cell proliferation through the inhibition of the ERK2 function. The present approach can be used broadly for modulating the cell signaling processes and understanding their roles in cellular function, as well as for the development of therapeutics.

Engineering of bacterial exotoxins for highly efficient and receptor-specific intracellular delivery of diverse cargos.

The intracellular delivery of proteins with high efficiency in a receptor-specific manner is of great significance in molecular medicine and biotechnology, but remains a challenge. Herein, we present the development of a highly efficient and receptor-specific delivery platform for protein cargos by combining the receptor binding domain of Escherichia coli Shiga-like toxin and the translocation domain of Pseudomonas aeruginosa exotoxin A. We demonstrated the utility and efficiency of the delivery platform by showing a cytosolic delivery of diverse proteins both in vitro and in vivo in a receptor-specific manner. In particular, the delivery system was shown to be effective for targeting an intracellular protein and consequently suppressing the tumor growth in xenograft mice. The present platform can be widely used for intracellular delivery of diverse functional macromolecules with high efficiency in a receptor-specific manner. Biotechnol. Bioeng. 2016;113: 1639-1646. © 2016 Wiley Periodicals, Inc.

Shiga-like toxin-based high-efficiency and receptor-specific intracellular delivery system for a protein.

The cell-specific cytosolic delivery of functional macromolecules with high efficiency is of great significance in molecular medicine and biotechnology. Herein, we present a Shiga-like toxin II-based high-efficiency and receptor-specific intracellular delivery system. We designed and constructed the Shiga-like toxin-based carrier (STC) to comprise the targeting and translocation domains, and used it for delivering a protein cargo. The STC was shown to deliver a protein cargo into the cytosol with high efficiency in a receptor-specific manner, exhibiting much higher efficiency than the most widely used cell-penetrating peptide. The general utility of the STC was demonstrated by modulating the targeting domain. The present delivery platform can be widely used for the intracellular delivery of diverse biomolecules in a receptor-specific and genetically encodable manner.

Oligomeric states of ASC specks regulate inflammatory responses by inflammasome in the extracellular space.

Inflammasomes are multi-protein complexes and play a crucial role in host defense against pathogens. Downstream inflammatory responses through inflammasomes are known to be related to the oligomerization degree of ASC specks, but the detailed mechanism still remains unexplored. Here, we demonstrate that oligomerization degrees of ASC specks regulate the caspase-1 activation in the extracellular space. A protein binder specific for a pyrin domain (PYD) of ASC (ASCPYD) was developed, and structural analysis revealed that the protein binder effectively inhibits the interaction between PYDs, disassembling ASC specks into low oligomeric states. ASC specks with a low oligomerization degree were shown to enhance the activation of caspase-1 by recruiting and processing more premature caspase-1 through interactions between CARD of caspase-1 (caspase-1CARD) and CARD of ASC (ASCCARD). These findings can provide insight into controlling the inflammasome-mediated inflammatory process as well as the development of inflammasome-targeting drugs.

Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms.

Bruton's tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the structural mechanism by which certain XLA mutations in the SH2 domain strongly perturb Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain required for Btk activation and to which multiple XLA mutations map. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevents activation of wild-type and TKI-resistant Btk, inhibiting Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.