Nov 29, 2023

In the upstream and downstream regions of the coding sequence of mRNA, there are non-coding regions known as untranslated regions (UTRs). Although these sequences do not code any part of the final protein translated from mRNA, they play a crucial role in the translation, degradation, and localization of mRNA.

In the sequence of the 5' UTR of mRNA, there is a structure called an upstream open reading frame (uORF). Approximately 50% of human genes have uORFs in their 5' UTRs. Due to the presence of start codons in their sequences, they are believed to interfere with the ribosome's determination of translation initiation, thus regulating the translation of the gene's "main open reading frame." However, the function of uORFs is not so straightforward. In recent years, researchers have found that some uORFs can be translated into peptides, and these peptides may have unexpected functions.

Lately, a research team from Sun Yat-sen University discovered that the translated product of the uORF of the RNF10 gene can serve as an antigenic epitope to support the development of a novel immunotherapy.

Unexpected Peptides

As the largest family of E3 ubiquitin ligases, the Ring Finger (RNF) family plays a crucial role in maintaining fundamental cellular functions. In various disease states, including cancer, dysregulation of RNF family members is not uncommon. Several RNF proteins are upregulated in the tumor environment, and the drug bortezomib targeting the ubiquitin-proteasome system has been on the market for some time. Unfortunately, there is still significant room for improvement in terms of specificity for bortezomib. Therefore, researchers are aiming to find a more specific anti-tumor drug by targeting the ubiquitin system.

To achieve this, a research team from Sun Yat-sen University screened the expression levels of RNF family members in the CT26 colon cancer cell line and found that a ubiquitin ligase named RNF10 was most abundant in CT26. In the 5' UTR of RNF10, the researchers identified a 261-nt uORF region. This sequence could be expressed in vitro, and antibodies recognizing this peptide (uPeptide) were able to detect a ten kDa protein in CT26 cells.

After injecting a 27-amino acid antigenic epitope targeting uPeptide into mice, the mice exhibited a robust and highly specific immune response. Simultaneously, RNF10 uPeptide significantly increased the expression of inflammatory factors in CD8+ T cells. These data collectively demonstrate the unexpected immunogenicity of RNF10 uPeptide.

The Anti-tumor Activity of RNF10 uPeptide

After detecting the immunogenicity, the researchers subsequently explored whether they could harness the immunogenicity of RNF10 uPeptide to develop it into an immunotherapeutic drug. To do this, the researchers cultured CD8+ T cells with dendritic cells that had been "activated" by the peptide. They then co-cultured these activated CD8+ T cells with CT26 tumor cells. The results showed that lymphocytes activated by the peptide exhibited potent cytotoxic activity against CT26 cells, effectively killing the tumor cells in vitro.

The anti-tumor activity of RNF10 uPeptide was validated in subsequent animal experiments. Mice that received the RNF10 uPeptide vaccine showed smaller tumor volumes after the transplantation of tumor cells. Further analysis of these mice revealed that those vaccinated with RNF10 uPeptide had more CD8+ T cells in their bodies, and these cells were capable of secreting more inflammatory factors, enhancing the immune system's ability to attack tumor cells. These data all demonstrate that RNF10 uPeptide can enhance the immune function in mice, alter the tumor microenvironment, and have a therapeutic effect on tumors.

From Mice to Humans

The RNF family, due to its extensive and essential physiological functions, has relatively conserved sequences throughout evolution. Since RNF10 uPeptide can have such a significant effect in mice, it is likely to have a similar role in human cells. To verify this theory, researchers investigated whether the human RNF10 gene could express a similar RNF10 uPeptide. By constructing the human RNF10 uORF sequence and transfecting it into human HEK293T cells, they successfully obtained human RNF10 uPeptide, demonstrating the human body's ability to produce RNF10 uPeptide. Furthermore, high levels of RNF10 expression were found in human pancreatic cancer tissues. Researchers also detected high levels of RNF10 uPeptide in pancreatic cancer cell lines. All these data further indicate the potential use of RNF10 uPeptide for immunotherapy in human cancers.

Subsequently, researchers constructed human RNF10 uPeptide and studied its anti-tumor activity in human tumors. Similar to what was observed in mouse cells, RNF10 uPeptide significantly increased the levels of inflammatory factors in T cells from pancreatic cancer patients, and these cells could also be expanded in vitro by RNF10 uPeptide. Moreover, these T cells exhibited strong cytotoxic effects against human tumor cells expressing RNF10 uPeptide. This evidence indicates the potential of RNF10 uPeptide as an immunotherapeutic agent.

To further validate the anti-tumor capability of RNF10 uPeptide, researchers used a patient-derived tumor xenograft (PDX) model. The results showed that when autologous dendritic cells and T cells carrying RNF10 uPeptide were injected into mice with cancer, the growth rate of their tumors was significantly lower compared to mice injected with dendritic cells and T cells that did not carry RNF10 uPeptide. Correspondingly, the former group of mice had a higher presence of RNF10 uPeptide-specific CD8+ T cells in their tumor tissues compared to the control group. These data once again suggest that RNF10 uPeptide might be an effective form of tumor vaccine.

Summary

While the study used a limited number of model tumor cells and lacked clinical data to further demonstrate the clinical relevance of RNF10 uPeptide, it is pioneering in discovering the anti-tumor activity associated with RNF10 uORF and the translated uPeptide. These findings help us reevaluate the functions of uORFs in both physiological and pathological contexts and provide new insights for the development of future immunotherapeutic drugs.

GenScript synthesized some of the antibodies and peptides used in this study.

Reference

[1] Zeng L, Zheng W, Zhang J, Wang J, Ji Q, Wu X, Meng Y, Zhu X, An epitope encoded by uORF of RNF10 elicits a therapeutic anti-tumor immune response, Molecular Therapy:Oncolytics (2023), doi: https://doi.org/10.1016/j.omto.2023.100737.