SUMMARY The complexity of liver tumorigenesis is underscored by the recently observed anti-oncogenic effects of oncoproteins, although the mechanisms are unclear. Shp2/Ptpn11 is a proto-oncogene in hematopoietic cells, and antagonizes the effect of tumor suppressor Pten in leukemogenesis. In contrast, we show here cooperative functions of Shp2 and Pten in suppressing hepatocarcinogenesis. Ablating both Shp2 and Pten in hepatocytes induced early-onset non-alcoholic steatohepatitis (NASH), and promoted genesis of liver tumor-initiating cells likely due to augmented cJun expression/activation, and elevated ROS and inflammation in the hepatic microenvironment. Inhibiting cJun partially suppressed NASH-driven liver tumorigenesis without improving NASH. SHP2 and PTEN deficiencies were detected in liver cancer patients with poor prognosis. These data depict a mechanism of hepato-oncogenesis and suggest a potential therapeutic strategy.

Overcoming tolerance to tumor-associated antigens remains a hurdle for cancer vaccine-based immunotherapy. A strategy to enhance the anti-tumor immune response is the inclusion of adjuvants to cancer vaccine protocols. In this report, we generated and systematically screened over twenty gene-based molecular adjuvants composed of cytokines, chemokines, and T cell co-stimulators for the ability to increase anti-tumor antigen T cell immunity. We identified several robust adjuvants whose addition to vaccine formulations resulted in enhanced T cell responses targeting the cancer antigens STEAP1 and TERT. We further characterized direct T cell stimulation through CD80-Fc and indirect T cell targeting via the dendritic cell activator Flt3L-Fc. Mechanistically, intramuscular delivery of Flt3L-Fc into mice was associated with a significant increase in infiltration of dendritic cells at the site of administration and trafficking of activated dendritic cells to the draining lymph node. Gene expression analysis of the muscle tissue confirmed a significant up-regulation in genes associated with dendritic cell signaling. Addition of CD80-Fc to STEAP1 vaccine formulation mimicked the engagement provided by DCs and increased T cell responses to STEAP1 by 8-fold, significantly increasing the frequency of antigen-specific cells expressing IFNγ, TNFα, and CD107a for both CD8 + and CD4 + T cells. CD80-Fc enhanced T cell responses to multiple tumor-associated antigens including Survivin and HPV, indicating its potential as a universal adjuvant for cancer vaccines. Together, the results of our study highlight the adjuvanting effect of T cell engagement either directly, CD80-Fc, or indirectly, Flt3L-Fc, for cancer vaccines.

Prostate cancer is the most common type of cancer in men. Immunotherapies such as Sipuleucel-T have shown that stimulating the immune system to target the prostate is a viable therapeutic option. Inovio is a clinical-stage biotechnology company that uses DNA vaccines as a novel immunotherapy strategy. An advantage to plasmid DNA vaccine therapy is the ability to encode adjuvants within the vaccine in order to increase immunogenicity and efficacy. We conducted an adjuvant screen in BALB/c mice with 25 candidate plasmid encoded genetic adjuvants in combination with the prostate cancer specific tumor-associated antigen STEAP1. Antigen-specific T cell responses were measured by IFNg ELISpot. The screen revealed that the addition of a plasmid encoding dendritic cell (DC)-activating Fms-like tyrosine kinase 3 ligand (Flt3L) fused to an Fc domain, significantly increased antigen-specific T cells (p<0.001, 2.8 fold). Mice treated with Flt3L-Fc had an enhanced immune response to STEAP1 vaccination as early as 7 days post dose 1 (p<0.01, 2.4 fold), which developed into an enhanced memory response measured 12 weeks later (p<0.001, 8.5 fold). Flow cytometry showed that Flt3L-Fc increased DC populations at the site of injection and at the draining lymph node 8 days following the initial vaccination. DCs are considered the most potent antigen-presenting cells in the immune system and DCs at the tumor site have been shown to be critical for T cell immunity. Our data shows that enhancing DC populations and function through the use of a genetic adjuvant can enhance the immunogenicity of a DNA cancer vaccine. Future studies will assess the efficacy of Flt3L-Fc in combination with STEAP1 in a mouse tumor model.