Regulation of Molecular Targets in Osteosarcoma Treatment.

The most prevalent malignant bone tumor, osteosarcoma, affects the growth plates of long bones in adolescents and young adults. Standard chemotherapeutic methods showed poor response rates in patients with recurrent and metastatic phases. Therefore, it is critical to develop novel and efficient targeted therapies to address relapse cases. In this regard, RNA interference technologies are encouraging options in cancer treatment, in which small interfering RNAs regulate the gene expression following RNA interference pathways. The determination of target tissue is as important as the selection of tissue-specific promoters. Moreover, small interfering RNAs should be delivered effectively into the cytoplasm. Lentiviral vectors could encapsulate and deliver the desired gene into the cell and integrate it into the genome, providing long-term regulation of targeted genes. Silencing overexpressed genes promote the tumor cells to lose invasiveness, prevents their proliferation, and triggers their apoptosis. The uniqueness of cancer cells among patients requires novel therapeutic methods that treat patients based on their unique mutations. Several studies showed the effectiveness of different approaches such as microRNA, drug- or chemotherapy-related methods in treating the disease; however, identifying various targets was challenging to understanding disease progression. In this regard, the patient-specific abnormal gene might be targeted using genomics and molecular advancements such as RNA interference approaches. Here, we review potential therapeutic targets for the RNA interference approach, which is applicable as a therapeutic option for osteosarcoma patients, and we point out how the small interfering RNA method becomes a promising approach for the unmet challenge.

Tumor-B-cell interactions promote isotype switching to an immunosuppressive IgG4 antibody response through upregulation of IL-10 in triple negative breast cancers.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive breast cancer for which there is currently no targeted therapy. Tumor-infiltrating B-cells (TIB) have been observed in tumor tissues of TNBC patients, but their functional role is unclear. IgG4 is one of four antibody subclasses of IgG expressed and secreted by B cells. Unlike other IgG isotypes, IgG4 has an immunosuppressive function and is induced by Th2-type cytokines. In cancers such as melanoma, IgG4 has been linked with advanced disease and poor patient survival. Therefore, we sought to determine if IgG4 + B cells are present and determine the mechanisms driving isotype switching in TNBC. METHODS: We performed co-culture assays to examine expression of Th2 cytokines by TNBC cells with and without the presence of B cells. We also performed in vitro class switching experiments with peripheral B cells with and without co-culture with TNBC cells in the presence or absence of an IL-10 blocking antibody. We examined expression of CD20+ TIB, IgG4 and Th2 cytokines by immunohistochemistry in 152 TNBC samples. Statistical analysis was done using Log-Rank and Cox-proportional hazards tests. RESULTS: Our findings indicate that B cells interact with TNBC to drive chronic inflammatory responses through increased expression of inflammatory cytokines including the TH2 cytokines IL-4 and IL-10. In vitro class switching studies show that interactions between TNBC cell lines and B cells drive isotype switching to the IgG4 isotype in an IL-10 dependent manner. In patient tissues, expression of IgG4 correlates with CD20 and tumor expression of IL-10. Both IgG4 and tumor IL-10 are associated to shorter recurrence free survival (RFS) and overall survival (OS) in TNBC. In a multi-variant analysis, IL-10 was associated with poor outcomes indicating that tumor IL-10 may drive immune escape. CONCLUSIONS: These findings indicate that interactions between TIB and TNBC results in activation of chronic inflammatory signals such as IL-10 and IL-4 that drive class switching to an IgG4 + subtype which may suppress antibody driven immune responses. The presence of IgG4 + B cells may serve as a biomarker for poor prognosis.

A Plant-Produced in vivo deglycosylated full-length Pfs48/45 as a Transmission-Blocking Vaccine Candidate against malaria.

Pfs48/45 is a leading antigen candidate for a transmission blocking (TB) vaccine. However, efforts to produce affordable, safe and correctly folded full-length Pfs48/45 using different protein expression systems have not produced an antigen with satisfactory TB activity. Pfs48/45 has 16 cysteines involved in disulfide bond formation, and the correct formation is critical for proper folding and induction of TB antibodies. Moreover, Pfs48/45 is not a glycoprotein in the native hosts, but contains potential glycosylation sites, which are aberrantly glycosylated during expression in eukaryotic systems. Here, we demonstrate for the first time that full length, Endo H in vivo enzymatic deglycosylated Pfs48/45 antigen is produced at a high level in plants and is structurally stable at elevated temperatures. Sera from mice immunized with this antigen showed strong inhibition in SMFA. Thus, Endo H in vivo enzymatic deglycosylated Pfs48/45 is a promising candidate for the development of an affordable TB vaccine, which may have the potential to save millions.

Engineering, and production of functionally active human Furin in N. benthamiana plant: In vivo post-translational processing of target proteins by Furin in plants.

A plant expression platform with eukaryotic post-translational modification (PTM) machinery has many advantages compared to other protein expression systems. This promising technology is useful for the production of a variety of recombinant proteins including, therapeutic proteins, vaccine antigens, native additives, and industrial enzymes. However, plants lack some of the important PTMs, including furin processing, which limits this system for the production of certain mammalian complex proteins of therapeutic value. Furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events. In this study, we engineered the human furin gene for expression in plants and demonstrated the production of a functional active recombinant truncated human furin in N. benthamiana plant. We demonstrate that plant produced human furin is highly active both in vivo and in vitro and specifically cleaved the tested target proteins, Factor IX (FIX) and Protective Antigen (PA83). We also demonstrate that both, enzymatic deglycosylation and proteolytic processing of target proteins can be achieved in vivo by co-expression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. It is highly expected that this strategy will have many potential applications in pharmaceutical industry and can be used to produce safe and affordable therapeutic proteins, antibodies, and vaccines using a plant expression system.

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-ß-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus.

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-ß-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes.