Prostaglandin E2 Induces miR675-5p to Promote Colorectal Tumor Metastasis via Modulation of p53 Expression.

BACKGROUND & AIMS: Prostaglandin E2 (PGE2) promotes colorectal tumor formation and progression by unknown mechanisms. We sought to identify microRNAs (miRNAs) that might mediate the effects of PGE2 on colorectal cancer (CRC) development. METHODS: We incubated LS174T colorectal cancer cells with PGE2 or without (control) and used miRNA-sequencing technology to compare expression patterns of miRNAs. We knocked down levels of specific miRNAs or proteins in cells using small interfering RNAs or genome editing. Cells were analyzed by immunoblot, quantitative polymerase chain reaction, chromosome immunoprecipitation, cell invasion, and luciferase reporter assays; we measured gene expression, binding activity, cell migration and invasion, and transcriptional activity of transcription factors. NOD-scidIL-2Rg-/- mice were given injections of LS174T cells, and growth of primary tumors and numbers of liver and lung metastases were quantified and analyzed by histology. We used public databases to identify correlations in gene expression pattern with patient outcomes. RESULTS: We identified miRNA 675-5p (miR675-5p) as the miRNA most highly up-regulated by incubation of colorectal cancer cells with PGE2. PGE2 increased expression of miR675-5p by activating expression of Myc, via activation of protein kinase B, also known as (AKT), nuclear factor κB, and ß-catenin. PGE2 increased the invasive activities of cultured CRC cells. LS174T cells incubated with PGE2 formed more liver and lung metastases in mice than control LS174T cells. We identified a 3' untranslated region in the TP53 messenger RNA that bound miR675-5p; binding resulted in loss of the p53 protein. Expression of miR675-5p or its precursor RNA, H19, correlated with expression of cyclooxygenase-1 and cyclooxygenase-2 and shorter survival times of patients with CRC. CONCLUSIONS: We found that treatment of mice with PGE2 increased CRC cells invasive activity and ability to form liver and lung metastases. PGE2 down-regulates expression of p53 by increasing expression of miR675-5p, which binds to and prevents translation of TP53 messenger RNA. These findings provide insight into the mechanisms by which PGE2 promotes tumor development and progression. Strategies to target PGE2 might be developed for treatment of CRC.

A novel platform to produce human monoclonal antibodies: The next generation of therapeutic human monoclonal antibodies discovery.

A new technology has been developed that allows human antibodies to be quickly generated against virtually any antigen. Using a novel process, naïve human B cells are isolated from tonsil tissue and transformed with efficiency up to 85%, thus utilizing a large portion of the human VDJ/VJ repertoire. Through ex vivo stimulation, the B cells class switch and may undergo somatic hypermutation, thus producing a human "library" of different IgG antibodies that can then be screened against any antigen. Since diversity is generated ex vivo, sampling immunized or previously exposed individuals is not necessary. Cells producing the antibody of interest can be isolated through limiting dilution cloning and the human antibody from the cells can be tested for biological activity. No humanization is necessary because the antibodies are produced from human B cells. By eliminating immunization and humanization steps, and screening a broadly diverse library, this platform should reduce both the cost and time involved in producing therapeutic monoclonal antibody candidates.

In vivo expansion, persistence, and function of peptide vaccine-induced CD8 T cells occur independently of CD4 T cells.

Significant efforts are being devoted toward the development of effective therapeutic vaccines against cancer. Specifically, well-characterized subunit vaccines, which are designed to generate antitumor cytotoxic CD8 T-cell responses. Because CD4 T cells participate at various stages of CD8 T-cell responses, it is important to study the role of CD4 T cells in the induction and persistence of antitumor CD8 T-cell responses by these vaccines. Recent evidence points to the requirement of CD4 T cells for the long-term persistence of memory CD8 T cells, which in the case of cancer immunotherapy would be critical for the prevention of tumor recurrences. The purpose of the present study was to assess whether CD4 T cells are necessary for the generation and maintenance of antigen-specific CD8 T cells induced by subunit (peptide or DNA) vaccines. We have used a vaccination strategy that combines synthetic peptides representing CD8 T-cell epitopes, a costimulatory anti-CD40 antibody and a Toll-like receptor agonist (TriVax) to generate large numbers of antigen-specific CD8 T-cell responses. Our results show that the rate of decline (clonal contraction) of the antigen-specific CD8 T cells and their functional state is not affected by the presence or absence of CD4 T cells throughout the immune response generated by TriVax. We believe that these results bear importance for the design of effective vaccination strategies against cancer.

Optimized DNA vaccines to specifically induce therapeutic CD8 T cell responses against autochthonous breast tumors.

BACKGROUND: Vaccines capable of inducing CD8 T cell responses to antigens expressed by tumor cells are considered as attractive choices for the treatment and prevention of malignant diseases. Our group has previously reported that immunization with synthetic peptide corresponding to a CD8 T cell epitope derived from the rat neu (rNEU) oncogene administered together with a Toll-like receptor agonist as adjuvant, induced immune responses that translated into prophylactic and therapeutic benefit against autochthonous tumors in an animal model of breast cancer (BALB-neuT mice). DNA-based vaccines offer some advantages over peptide vaccines, such as the possibility of including multiple CD8 T cell epitopes in a single construct. MATERIALS AND METHODS: Plasmids encoding a fragment of rNEU were designed to elicit CD8 T cell responses but no antibody responses. We evaluated the use of the modified plasmids as DNA vaccines for their ability to generate effective CD8 T cell responses against breast tumors expressing rNEU. RESULTS: DNA-based vaccines using modified plasmids were very effective in specifically stimulating tumor-reactive CD8 T cell responses. Moreover, vaccination with the modified DNA plasmids resulted in significant anti-tumor effects that were mediated by CD8 T cells without the requirement of generating antibodies to the product of rNEU. CONCLUSIONS: DNA vaccination is a viable alternative to peptide vaccination to induce potent anti-tumor CD8 T cell responses that provide effective therapeutic benefit. These results bear importance for the design of DNA vaccines for the treatment and prevention of cancer.