Correction: Generation of novel affibody molecules targeting the EBV LMP2A N-terminal domain with inhibiting effects on the proliferation of nasopharyngeal carcinoma cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Generation of novel affibody molecules targeting the EBV LMP2A N-terminal domain with inhibiting effects on the proliferation of nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) induced by latent infection with Epstein-Barr virus (EBV) remains the most common head and neck cancer in Southeast Asia, especially in the southern part of China. It is well known that persistent expression of two EBV latent membrane proteins (LMP1/LMP2A) plays a key role in nasopharyngeal carcinogenesis. Therefore, the therapeutic approach of targeting the LMP1/LMP2A protein and subsequently blocking the LMP1/LMP2A-mediated signalling pathway has been considered for treating patients with NPC. Recently, affibody molecules, a new class of small (~6.5 kDa) affinity proteins, have been confirmed to be powerful generalisable tools for developing imaging or therapeutic agents by targeting specific molecules. In this study, three EBV LMP2A N-terminal domain-binding affibody molecules (ZLMP2A-N85, ZLMP2A-N110 and ZLMP2A-N252) were identified by screening a phage-displayed peptide library, and their high affinity and specificity for the EBV LMP2A N-terminal domain were confirmed by surface plasmon resonance (SPR), indirect immunofluorescence, co-immunoprecipitation and near-infrared small animal fluorescence imaging in vitro and in vivo. Moreover, affibody molecules targeting the EBV LMP2A N-terminal domain significantly reduced the viability of the EBV-positive cell lines C666-1, CNE-2Z and B95-8. Further investigations showed that affibody ZLMP2A-N110 could inhibit the phosphorylation of AKT, GSK-3ß and ß-catenin signalling proteins, leading to suppression of ß-catenin nuclear translocation and subsequent inhibition of c-Myc oncogene expression, which may be responsible for the reduced viability of NPC-derived cell lines. In conclusion, our findings provide a strong evidence that three novel EBV LMP2A N-terminal domain-binding affibody molecules have great potential for utilisation and development as agents for both molecular imaging and targeted therapy of EBV-related NPC.

HPV16 E7-impaired keratinocyte differentiation leads to tumorigenesis via cell cycle/pRb/involucrin/spectrin/adducin cascade.

Here, we used codon usage technology to generate two codon-modified human papillomavirus (HPV)16 E7 genes and, together with wild-type E7, to construct three HPV16 E7 gene plasmids: Wt-E7, HB1-E7, and HB2-E7. The three HPV 16 E7 plasmids were used to investigate how HPV16 E7 protein was expressed in different cells and how this oncoprotein deregulated cellular and molecular events in human keratinocytes to induce carcinogenesis. We discovered that codon usage of HPV16 E7 gene played a key role in determining expression of E7 oncoprotein in all tested cells. HPV16 E7 inhibited significantly expression of pRb to impair keratinocyte differentiation and disrupted development of skin epidermis in mice. HPV16 E7 increased substantially the number of G0/G1 cells associated with upregulation of cyclin D2 and downregulation of cyclin B1 in keratinocytes. HPV16 E7 not only inhibited expression of involucrin and α-spectrin but also disrupted the organization of involucrin filaments and spectrin cytoskeleton. Furthermore, HPV16 E7 inhibited expression of ß-adducin, destroyed its cytoskeletal structure and induced phosphorylation of ß-adducin(Ser662) in keratinocytes. Importantly, HPV16 E7 induced carcinogenesis in mice associated with expression of phosphorylated ß-adducin(Ser662) and its nucleus-translocation. In conclusion, we provided evidence that HPV16 E7 oncoprotein inhibited keratinocyte differentiation in vitro and in vivo leading to carcinogenesis through cell cycle arrest and disruption of pRb/involucrin/spectrin/adducin cascade.

Novel EBV LMP-2-affibody and affitoxin in molecular imaging and targeted therapy of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) infection is closely linked to several human malignancies including endemic Burkitt's lymphoma, Hodgkin's lymphoma and nasopharyngeal carcinomas (NPC). Latent membrane protein 2 (LMP-2) of EBV plays a pivotal role in pathogenesis of EBV-related tumors and thus, is a potential target for diagnosis and targeted therapy of EBV LMP-2+ malignant cancers. Affibody molecules are developing as imaging probes and tumor-targeted delivery of small molecules. In this study, four EBV LMP-2-binding affibodies (ZEBV LMP-212, ZEBV LMP-2132, ZEBV LMP-2137, and ZEBV LMP-2142) were identified by screening a phage-displayed LMP-2 peptide library for molecular imaging and targeted therapy in EBV xenograft mice model. ZEBV LMP-2 affibody has high binding affinity for EBV LMP-2 and accumulates in mouse tumor derived from EBV LMP-2+ xenografts for 24 h after intravenous (IV) injection. Subsequent fusion of Pseudomonas exotoxin PE38KDEL to the ZEBV LMP-2 142 affibody led to production of Z142X affitoxin. This fused Z142X affitoxin exhibits high cytotoxicity specific for EBV+ cells in vitro and significant antitumor effect in mice bearing EBV+ tumor xenografts by IV injection. The data provide the proof of principle that EBV LMP-2-speicifc affibody molecules are useful for molecular imaging diagnosis and have potentials for targeted therapy of LMP-2-expressing EBV malignancies.