Discussion

In this study, we explored the functional role, molecular mechanism, and clinical significance of psiTPTE22-HERV in gastric cancer. Consistently down-regulated across various tumor types, psiTPTE22-HERV plays an important role in suppressing the development and metastasis of gastric cancer. Its down-regulation in tumors is mediated by promoter methylation and serves as an independent risk factor for poor prognosis in gastric cancer patients. Notably, psiTPTE22-HERV exerted tumor-suppressive functions, at least partially, by down-regulating EIF4E via the PI3K/AKT/mTOR signaling pathway.

We demonstrated the tumor-suppressive function of psiTPTE22-HERV in gastric cancer both in vitro and in vivo. Using both gain- and loss-of-function assays, we found that psiTPTE22-HERV significantly suppressed gastric cancer cell growth by reducing cell viability and colony formation ability, inhibiting G1–S cell cycle transition, and inducing apoptosis. In vivo tumorigenesis assay further confirmed the tumor-suppressive role of psiTPTE22-HERV in suppressing the growth of subcutaneous xenografts. Supporting our clinical data, which showed that psiTPTE22-HERV down-regulation was correlated with tumor metastasis, we observed that psiTPTE22-HERV inhibited the migration and invasion of cancer cells in vitro and suppressed their metastasis to distant organs in vivo.

The molecular mechanism underlying the tumor-suppressive function of psiTPTE22-HERV may be associated with the suppression of protein synthesis with regard to the down-regulation of protein synthesis-related genes. Protein synthesis is essential for oncogenic cellular transformation.24 Specifically, we identified and validated two genes directly involved in protein synthesis initiation, EIF4EBP1 and EIF4E, which were down-regulated by psiTPTE22-HERV. The reverse regulation of EIF4EBP1 and EIF4E by psiTPTE22-HERV was demonstrated by the ectopic expression and knockdown of psiTPTE22-HERV in gastric cancer cells. Significant inverse correlations between psiTPTE22-HERV and both EIF4EBP1 and EIF4E were observed in primary gastric tumors. These results provide solid evidence supporting the effects of psiTPTE22-HERV on EIF4EBP1 and EIF4E, although these effects may not be direct but rather mediated through the suppression of the PI3K/AKT/mTOR pathway. The PI3K/AKT/mTOR pathway is known to be activated in gastric cancer, with accumulating evidence linking it to cell growth, metabolism, survival, metastasis, and resistance to chemotherapy.25 Increased EIF4E expression has been reported in gastric cancer, contributing to cell proliferation and disease progression.26 Moreover, overexpression of EIF4E is associated with tumor vascular invasion and lower survival rates in patients with gastric cancer.27 Phosphorylation of EIF4EBP1 can lead to the release of EIF4E, thereby relieving translational repression and enhancing oncogenic protein synthesis. However, our results showed that psiTPTE22-HERV decreased the levels of EIF4E, EIF4EBP1, and p-EIF4EBP1. The reduction in EIF4EBP1 levels may result from decreased EIF4E levels and contribute to lower p-EIF4EBP1 levels. Ultimately, the decrease in the oncogene EIF4E may significantly mediate the tumor-suppressive function of psiTPTE22-HERV.

Cytoplasmic and nuclear EIF4E play distinct roles: cytoplasmic EIF4E is essential for initiating protein synthesis, while nuclear EIF4E facilitates mRNA export.23 In our study, only cytoplasmic EIF4E levels were altered following psiTPTE22-HERV overexpression or knockdown, indicating that psiTPTE22-HERV primarily influences the regulation of protein synthesis. Hyperactive AKT can induce proteotoxic stress,28 characterized by the accumulation of misfolded or unfolded proteins. Additionally, mTOR is essential for the proteotoxic stress response.29 Notably, we observed that the expression of psiTPTE22-HERV led to the suppression of both AKT and mTOR. These findings suggest that psiTPTE22-HERV may help reduce cellular stress, ultimately resulting in decreased EIF4E expression.

While we have established that psiTPTE22-HERV is primarily regulated by promoter methylation at specific CpG sites, the specific transcription factors and binding motifs involved require further characterization. The molecular mechanism underlying its regulation of the PI3K/AKT/mTOR/EIF4E axis also requires further investigation. A major technical challenge in studying this gene is the lack of reliable antibodies. We made multiple attempts to generate antibodies using full-length proteins and synthetic peptides based on epitope prediction as immunogens, but the resulting antibodies were not satisfactory in terms of specificity and affinity. This limitation may hinder the characterization of its direct interacting molecules. Future efforts are needed to develop a specific and effective antibody for clinical applications, such as immunohistochemistry staining. Given its consistent down-regulation in various cancer types, the functional mechanisms and clinical implications of psiTPTE22-HERV in other cancers also merit exploration.