Using Epstein-Barr virus (EBV)-induced cancer cells and HeLa cells as a comparative study model, a novel and safe dual-EBV-oncoproteins-targeting pH-responsive peptide engineering, coating, and guiding approach to achieve precision targeting and treatment strategy against EBV-associated cancers is introduced. Individual functional peptide sequences that specifically bind to two overexpressed EBV-specific oncoproteins, EBNA1 (a latent cellular protein) and LMP1 (a transmembrane protein), are engineered in three different ways and incorporated with a pH-sensitive tumor microenvironment (TME)-cleavable linker onto the upconversion nanoparticles (UCNP) NaGdF 4 :Yb 3+ , Er 3+ @NaGdF 4 (UCNP-P n , n = 5, 6, and 7). A synergistic combination of the transmembrane LMP1 targeting ability and the pH responsiveness of UCNP-P n is found to give specific cancer differentiation with higher cellular uptake and accumulation in EBV-infected cells, thus a lower dose is needed and the side effects and health risks from treatment would be greatly reduced. It also gives responsive UC signal enhancement upon targeted dual-protein binding and shows efficacious EBV cancer inhibition in vitro and in vivo. This is the first example of simultaneous imaging and inhibition of two EBV latent proteins, and serves as a blueprint for next-generation peptide-guided precision delivery nanosystem for the safe monitoring and treatment against one specific cancer.

Epstein–Barr nuclear antigen 1 (EBNA1) plays a vital role in the maintenance of the viral genome and is the only viral protein expressed in nearly all forms of Epstein–Barr virus (EBV) latency and EBV-associated diseases, including numerous cancer types. To our knowledge, no specific agent against EBV genes or proteins has been established to target EBV lytic reactivation. Here we report an EBNA1- and Zn2+-responsive probe (ZRL5P4) which alone could reactivate the EBV lytic cycle through specific disruption of EBNA1. We have utilized the Zn2+chelator to further interfere with the higher order of EBNA1 self-association. The bioprobe ZRL5P4can respond independently to its interactions with Zn2+and EBNA1 with different fluorescence changes. It can selectively enter the nuclei of EBV-positive cells and disrupt the oligomerization andoriP-enhanced transactivation of EBNA1. ZRL5P4can also specifically enhance Dicer1 and PML expression, molecular events which had been reported to occur after the depletion of EBNA1 expression. Importantly, we found that treatment with ZRL5P4alone could reactivate EBV lytic induction by expressing the early and late EBV lytic genes/proteins. Lytic induction is likely mediated by disruption of EBNA1 oligomerization and the subsequent change of Dicer1 expression. Our probe ZRL5P4is an EBV protein-specific agent that potently reactivates EBV from latency, leading to the shrinkage of EBV-positive tumors, and our study also suggests the association of EBNA1 oligomerization with the maintenance of EBV latency.

We and others have previously shown that the canonical nuclear factor kappa-B (NF-κB) pathway is essential to nasopharyngeal carcinoma (NPC) tumor development and angiogenesis, suggesting that the NF-κB pathway, including its upstream modulators and downstream effectors, are potential therapeutic targets for NPC. The inhibitor of upstream IκB kinase (IKK), PS1145, is a small molecule which can specifically inhibit the IκB phosphorylation and degradation and the subsequent nuclear translocation of NF-κB. The present study aims to determine the anti-tumor activity of PS1145 on NPC. Our results showed that PS1145 significantly inhibited the growth of tumorigenic NPC cell lines, but not in the normal nasopharyngeal epithelial cell line. Results in the in vivo study showed that low concentration of PS1145 (3 mg/kg) could significantly suppress the subcutaneous tumor formation in the nude mice bearing NPC xenografts. Apparent adverse effects were not observed in the animal study. Drug resistance against PS1145 seems to be associated with the increased levels of active NF-kB p65 and change of expression levels of kruppel-like factor 4. As can be seen, PS1145 appears to be a safe agent for animal experiments and its effects are tumor-specific, and the proteins associated with the drug resistance of PS1145 are implied.

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV)-associated malignancy ranking as the 23rd most common cancer globally, while its incidence rate ranked the 9th in southeast Asia. Tumor metastasis is the dominant cause for treatment failure in NPC and metastatic NPC is yet incurable. The Wnt/β-catenin signaling pathway plays an important role in many processes such as cell proliferation, differentiation, epithelial–mesenchymal transition (EMT), and self-renewal of stem cells and cancer stem cells (CSCs). Both the EMT process and CSCs are believed to play a critical role in cancer metastasis. We here investigated whether the specific CBP/β-catenin Wnt antagonist, IGC-001, affects the metastasis of NPC cells. We found that ICG-001 treatment could reduce the adhesion capability of NPC cells to extracellular matrix and to capillary endothelial cells and reduce the tumor cell migration and invasion, events which are closely associated with distant metastasis. Through a screening of EMT and CSC-related microRNAs, it was found that miR-134 was consistently upregulated by ICG-001 treatment in NPC cells. Very few reports have mentioned the functional role of miR-134 in NPC, except that the expression was found to be downregulated in NPC. Transient transfection of miR-134 into NPC cells reduced their cell adhesion, migration, and invasion capability, but did not affect the growth of CSC-enriched tumor spheres. Subsequently, we found that the ICG-001-induced miR-134 expression resulting in downregulation of integrin β1 (ITGB1). Such downregulation reduced cell adhesion and migration capability, as demonstrated by siRNA-mediated knockdown of ITGB1. Direct targeting of ITGB1 by miR-134 was confirmed by the 3′-UTR luciferase assay. Lastly, using an in vivo lung metastasis assay, we showed that ICG-001 transient overexpression of miR-134 or stable overexpression of miR-134 could significantly reduce the lung metastasis of NPC cells. Taken together, we present here evidence that modulation of Wnt/β-catenin signaling pathway could inhibit the metastasis of NPC through the miR-134/ITGB1 axis.

Fig. 6. Production of infectious EBV particle in response to ZRL 5 P 4 . The HONE-1-EBV cell line, which expresses GFP to indicate the presence of the EBV genome, was used. This cell line was treated with 10 μM ZRL 5 P 2 or ZRL 5 P 4 for 4 d, and the viral particles released in the culture medium were detected by the Raji cell assay. The culture medium was added to Raji cells for 3 d, and GFP expression reflects the reinfection by the HONE-1-released EBV particles. (A) Representative results are shown. The GFP signal was detected by UV light exposure, and the cell morphology was captured by phase-contrast light microscopy and the bright-field image was merged with the GFP image. Magnification, 400×. (Scale bars, 100 μm.) (B) Relative average viral titer in response to ZRL 5 P 2 or ZRL 5 P 4 was compared with the solvent control (DMSO). The Raji cell assay was performed in triplicate for each treatment. **P < 0.01, statistically significant difference. Data are expressed as the means ± SD. (C) Representative images of immunofluorescent analysis of Dicer1 and PML in HONE-1-EBV cells in response to 10 μM ZRL 5 P 2 or ZRL 5 P 4 . The nuclei were counterstained with DAPI and indicated in blue. (Scale bars, 50 μm.) (D) Comparison of the number of Zta-positive cells after treatment with ZRL 5 P 4 in the presence versus the absence of Dicer1. HONE-1-EBV and NPC43 were included. Gene silencing of Dicer1 was achieved by siRNA transfection. Zta was detected by immunofluorescent analysis. Control siRNA (siCTL) was used as a negative control. Relative percentage of Zta-positive was compared against the DMSO solvent control in the siRNA control cells. *P < 0.05. 5542