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Transfection is based on nonviral delivery of nucleic acids or proteins into cells. Viral approaches are being used; nevertheless, their translational capacity is nowadays decreasing due to persistent fear of their safety, therefore creating space for the field of nanotechnology. However, nanomedical approaches introducing static nanoparticles for the delivery of biologically active molecules are very likely to be overshadowed by the vast potential of nanorobotics. We hereby present a rapid nonviral transfection of protein into a difficult-to-transfect prostate cancer cell line facilitated by chemically powered rectangular virus-sized (68 nm × 33 nm) nanorobots. The enhanced diffusion of these biocompatible nanorobots is the key to their fast internalization into cells, happening in a matter of minutes and being up to 6-fold more efficient compared to static nanorobots in a nonfueled environment. The Au/Ag plasmonic nature of these nanorobots makes them simply traceable and allows for their detailed subcellular localization. Protein transfection mediated by such nanorobots is an important step forward, challenging the field of nanomedicine and having potential in future translational medical research.

Inhibitors of DNA methyltransferases (DNMTis) or histone deacetylases (HDACis) are epigenetic drugs which are investigated since decades. Several have been approved and are applied in the treatment of hematopoietic and lymphatic malignancies, although their mode of action has not been fully understood. Two recent findings improved mechanistic insights: i) activation of human endogenous retroviral elements (HERVs) with concomitant synthesis of double-stranded RNAs (dsRNAs), and ii) massive activation of promoters from long terminal repeats (LTRs) which originated from past HERV invasions. These dsRNAs activate an antiviral response pathway followed by apoptosis. LTR promoter activation leads to synthesis of non-annotated transcripts potentially encoding novel or cryptic proteins. Here, we discuss the current knowledge of the molecular effects exerted by epigenetic drugs with a focus on DNMTis and HDACis. We highlight the role in LTR activation and provide novel data from both in vitro and in vivo epigenetic drug treatment.

Metals are known for playing essential roles in human physiology. Copper and zinc are trace elements closely dependent on one another and are involved in cell proliferation, growth, gene expression, apoptosis and other processes. Their homeostasis is crucial and tightly controlled by a resourceful system of transporters and transport proteins which deliver copper and zinc ions to their target sites. Abnormal zinc and copper homeostasis can be seen in a number of malignancies and also in head and neck cancer. Imbalance in this homeostasis is observed as an elevation or decrease of copper and zinc ions in serum or tissue levels in patients with cancer. In head and neck cancer these altered levels stand out from those of other malignancies which makes them an object of interest and therefore zinc and copper ions might be a good target for further research of head and neck cancer development and progression. This review aims to summarize the physiological roles of copper and zinc, its binding and transport mechanisms, and based on those, its role in head and neck cancer. To provide stronger evidence, dysregulation of levels is analysed by a meta-analytical approach.

Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.

Epigenetic drugs are currently used for the treatment of several hematologic malignancies, but their mechanism of action remains poorly understood. By using a previously described reporter cell line for epigenetic reactivation of the DAPK1 locus, we have shown that epigenetic treatment causes transcription from uncharacterized intronic transcription start sites (TSSs), thereby generating DAPK1 mRNA with novel first exons. Based on these findings, we analyzed whether inhibition of DNA-Methyltransferases (DNMTs), Histone deacetylases (HDACs), or both resulted in the genome-wide induction of non-canonical TSSs. While epigenetic treatment altered expression of known promoter sites, we observed that both HDAC- and DNMT-inhibitors predominantly induced de novo transcription from cryptic promoters encoded in long-terminal repeat (LTR) retrotransposons. These LTR-associated 'treatment induced, not-annotated TSS' (TINATs) are currently not annotated and normally silenced in almost all cell types with the exception of testicular und thymic tissue. In the majority of cases, these TINATs arose most commonly from LTR12 elements, particularly LTR12C (which apparently provides 50% of all TINATs). TINAT activation after DNMT-inhibitors (DNMTi) coincided with DNA hypomethylation and gain in H3K4me3, H3K9ac, and H3K27ac histone marks. In contrast, HDAC-inhibitors (HDACi) induced only canonical TSSs in association with histone acetylation, but TINATs via a yet unknown mechanism. Nevertheless, both inhibitors convergently induced unidirectional transcription from identical TINAT sites. Moreover, we found a consensus GATA2 binding motif which strongly distinguished LTR12Cs with TINATs from LTR12Cs without TINATs, supporting that GATA2 is likely the upstream transcription factor responsible for TINAT activation. TINATs originating from non-canonical TSSs located within introns of protein-coding genes frequently spliced into downstream exons thereby creating LTR/non-LTR fusion transcripts that harbor novel in place of canonical exon sequence at their 5' end. The resulting transcripts encode truncated or chimeric open reading frames which translated into currently uncharacterized protein isoforms with predicted abnormal functions or immunogenic potential, the last one based on their foreign sequence and capability of being presented on MHC-class I molecules. In summary, we could show that DNMTi and/or HDACi do not predominantly alter the expression of canonical genes, but induce de novo transcription of LTRs especially of the LTR12 family, resulting in numerous fusion transcripts that encode novel protein isoforms which might have the potential to influence cell proliferation or might be an elegant explanation for the priming effect of epigenetic therapy. Ongoing experiments are investigating the functional mechanisms of TINAT reactivation upon epigenetic drug treatment and future proteomic approaches combined with T-cell cytotoxicity assays will further shed light on the interaction between epigenetic and immune therapy and the role of ERV-derived antigen presentation. Disclosures Lübbert: Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding.