The transcription factor Snail2 is a repressor of E-cadherin expression during carcinogenesis; however, the specific mechanisms involved in this process in human colorectal cancer (CRC) remain largely unknown. We checked the expression of Snail2 in several clinical CRC specimens. Then, we established Snail2-overexpressing and knockdown cell lines to determine the function of Snail2 during EMT and metastasis processes in CRC. In addition, we used luciferase reporter assay to explore how Snail2 inhibits the expression of E-cadherin and induces EMT. We found that the expression of Snail2 was higher in clinical specimens of colorectal cancer (CRC) compared to non-cancerous tissues. Overexpression of Snail2 induced migration and metastatic properties in CRC cells in vitro and in vivo. Furthermore, overexpression of Snail2 promoted the occurrence of the epithelial–mesenchymal transition (EMT), downregulating the expression of E-cadherin and upregulating that of vimentin. Specifically, Snail2 could interact with HDAC6 and then recruited HDAC6 and PRC2 to the promoter of E-cadherin and thus inhibited the expression of E-cadherin, promoting EMT and inducing invasion and metastasis of CRC. Our study reveals that Snail2 might epigenetically suppress the expression of E-cadherin during CRC metastasis.

The tumor microenvironment (TME) provides necessary nutrition for tumor growth and recruits immunosuppressive factors including regulatory T cells and myeloid-derived suppressor cells (MDSCs) to inhibit the anti-tumor immune response induced by immunotherapy. As a main TME component, cancer associated fibroblasts (CAFs) can restrain T cell infiltration and activity through extracellular matrix remodeling. Vaccines targeting fibroblast-activating protein α (FAPα), which is mainly expressed on the CAF surface, can eliminate CAFs in tumors and regulate the TME, enhancing the potency of T cell-mediated anti-tumor effects. However, the anti-tumor effects were not fully realized as the tumor induces a large number of peripheral MDSCs during its growth, rendering the body of mice in an immunosuppressive state and preventing the vaccine from inducing effective anti-tumor immune responses. Here, we developed a dual-targeted DNA vaccine OsFS, targeting tumor matrix antigen FAPα and tumor cell antigen survivin simultaneously, exhibited enhanced antineoplastic effects in an established breast cancer model. Moreover, doxorubicin (Dox) pretreatment to remove the peripheral MDSCs induced to regulate the peripheral immune environment could further facilitate the anti-tumor activity of the vaccine. These results indicated that combination treatment of the tumor cells and the TME dual-targeting vaccine plus Dox could effectively realize the anti-tumor activity of the vaccine by decreasing immunosuppressive factors and inducing more tumor-infiltrating lymphocytes, which may offer important guidance for clinical research regarding the combination of the DNA vaccine with low-dose Dox.

Adeno-associated virus (AAV) vectors have been utilized extensively in gene therapy and gene function studies, as strong transgene expression is a prerequisite for positive outcomes. AAV8 was reported as the most efficient AAV serotype for transduction of the liver, brain and muscle compared with other serotypes. However, AAV8-mediated transduction of human hepatocytes is rather poor with approximately 20-fold lower efficiency compared with that of mouse hepatocytes. Therefore, we applied the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance AAV8-mediated transgene expression driven by a combination promoter (CAG promoter) with a CMV-IE enhancer and chicken beta-actin promoter for a more efficient viral vector. Transgene expression from recombinant AAV8 (rAAV8) vectors harboring a red fluorescent protein (RFP) reporter gene with or without WPRE were evaluated in vitro and in vivo. The results demonstrated that WPRE improved AAV8-mediated RFP expression in different cell lines with clear increases of transgene expression in the liver, brain or muscle of animals. The findings of this study will help to substantially reduce the quantity of viral particles that must be injected in order to reach a therapeutic level of transgene expression in gene therapy. Consequently, such dose reductions may lessen the potential risks associated with high doses of viral vectors.