Endogenous retroviruses (ERVs) are derived from past exogenous retroviral infections of germ cells, which integrated into the genome of mammals and are inherited in a Mendelian manner. ERVs belong to the class of retro-elements possessing three main viral genes: group-specific antigen (gag), polymerase (pol) and envelope (env) flanked by two long terminal repeats (LTR). More than 8% of the human genome is considered of retroviral origin. Most ERV genes are protein non-functional due to recombination, mutations and deletions; however some ERV gag, pol and env genes produce functional proteins. Although largely gene defective, the LTRs of ERVs retained their regulatory activities resulting in high transcriptional levels; and in addition represent most of the ~10,000 annotated long non-coding (lnc) RNAs. In somatic cells ERVs are generally silent; however we and others demonstrated ERVs become activated during tumorigenesis.
Recently we showed that induction of immune responses in human cancer cells by DNA methyltransferase inhibitors (DNMTi), like 5-azacytidine (Aza) and 2'-deoxy-5-azacytidine (Dac) is primarily due to the activation of (hyper)-methylated ERVs (Chiappinelli et al. 2015, Cell 162: 974-86). Furthermore, the unique genomic architecture of ERVs with 5 and 3 LTRs induced sense and antisense RNAs leading to formation of cytosolic dsRNA, which activated the cellular viral RNA sensing and defense pathway (like TLR3 and MAVS) triggering the type I interferon (IFN) response. This kind of response following ERV activation is called viral mimicry or endogenous viremia. Using ovarian carcinoma (OVCA) cell lines as a model, we showed that Aza significantly induced multiple ERV families and IFN stimulated genes (ISG). The induction was due to the formation of dsRNA, which was abolished with RNAse III (hydrolyzing dsRNA) and TLR3 or MAVS specific inhibition. ISGs were also directly induced by transfected ERV genes. Subsequently, a cohort of primary human serous OVCA tissues was analyzed and a significant positive correlation between ERV and ISG expression could be demonstrated. We and others previously showed that treatment with the DNMTi Aza positively contributed to immune checkpoint blockade therapy in a mouse model and in human cancers. Like exogenous retroviruses, which upregulate immune checkpoint genes (ICG), we also evaluated for a possible connection between ERV induction and ICG expression in primary tumors. Presently, we demonstrate that human serous OVCA tissues express significantly higher ICGs than control ovarian tissues, with VTCN-1 (B7-H4) as the highest expressed ICG. Using serous OVCA cell lines transfected with different ERV genes we could show an early induction of ICGs, primarily LGALS9 and PD-L1. Further analysis of tumor infiltrating immune cells using the same serous OVCA cohort and multiplex immunohistochemistry (IHC), we detect in some tumors high levels of tumor-associated CD79a, CD3+/CD8- and CD3+/CD8+ cells. Moreover, we find a direct link between ISG, ICG and infiltrating immune cells in OVCA. Analyzing other human tumors, e.g. brain tumors, like glioblastoma multiforme (GBM) and giant cell GBM (gcGBM) also demonstrate a significant correlation between high ERV induction and ISG as well as ICG expression. In contrast to serous OVCA, PD1 and VTCN-1 were the highest expressed ICG in GBM and gcGBM, which could also be shown with ERV transfected GBM cell lines. Using IHC gcGBM shows a high level of immune cell infiltration, which associated with giant cells and ICG expression, like PD-L1. Interestingly, the highest ERV expression was found in gcGBM, which correlated with endomitosis and cell fusion leading to giant nuclei and multinucleated cells as also shown with a stable ERV transfected GBM cell line.
In summary, DNMTi mediated loss of methylation induced ERVs forming dsRNA in tumor cells and stimulated the innate immune system via ISGs. ERV RNA per se induced ISGs, but also ICGs similar to exogenous retroviruses in order to escape the immune system. Different upregulated ERVs, ISGs and ICGs were found in different cancers, like serous OVCA, GBM and gcGBM indicating diverse tumor and tumor microenvironment signatures. Thus ERVs have an ambivalent role in tumorigenesis, where tumor microenvironmental factors tip the balance being detrimental by generating immune evasion or beneficial for cancer therapy by stimulating the innate immune system.
Citation Format: Reiner Strick. Functional role of endogenous retroviruses in cancer immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr IA02.