Results

psiTPTE22-HERV is commonly down-regulated in primary tumors across various cancer types

To comprehensively evaluate psiTPTE22-HERV expression in cancers, we analyzed the TCGA data. Among the 15 cancer types with both tumor and adjacent non-tumor tissues, psiTPTE22-HERV was significantly down-regulated in tumors compared with non-tumor tissues in 14 types, including gastric cancer (Fig. 1A). The exception was kidney renal papillary cell carcinoma, which showed no substantial down-regulation. Most non-tumor tissues displayed ubiquitous psiTPTE22-HERV expression, with 12 out of 15 types showing similar or higher levels relative to all non-tumor tissues (Fig. 1B). Reverse transcription PCR confirmed psiTPTE22-HERV expression in various normal adult tissues, including the normal stomach (Fig. 1C), whereas its expression was not detected in 76% (19/25) of the cancer cell lines (Fig. 1D). These findings demonstrate that psiTPTE22-HERV is expressed in most normal tissues and is consistently down-regulated across a wide spectrum of cancer types.

Figure 1: Down-regulation of psiTPTE22-HERV in tumor tissues across multiple cancer types. (A) Significant down-regulation of psiTPTE22-HERV in primary tumors versus matched non-tumor tissues in 14/15 cancer types. (B) Robust psiTPTE22-HERV expression in non-tumor tissues. (C, D)psiTPTE22-HERV expression in normal adult tissues, including stomach mucosa, and its silencing/down-regulation in cancer cell lines as indicated by PCR. The data were presented as median (interquartile range) (A) or box-and-whiskers (Tukey) (B) and compared using Mann–Whitney U test (A) or Kruskal–Wallis test (B; versus all data). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

psiTPTE22-HERV is down-regulated by promoter methylation in gastric cancer cells and primary tumors

We further investigated the expression and regulatory mechanism of psiTPTE22-HERV in gastric cancer. psiTPTE22-HERV was silenced in the majority of the tested gastric cancer cell lines (9/10, 90%) (Fig. 2A), but its expression was restored following treatment with the DNA demethylation agent 5-Aza (Fig. 2B). The bisulfite genomic sequencing results demonstrated that cells with down-regulated or silenced psiTPTE22-HERV exhibited significantly higher methylation levels at 11 out of the 26 CpG sites analyzed than cells with psiTPTE22-HERV expression or restoration (P < 0.05; Fig. 2C). The PCR analyses revealed that psiTPTE22-HERV expression was significantly down-regulated in gastric tumors compared with paired adjacent non-tumor tissues in Chinese patients (n = 40; P = 0.001; Fig. 2D1). This finding was corroborated by RNA-sequencing data from TCGA (n = 32; P < 0.0001; Fig. 2D2). Furthermore, psiTPTE22-HERV expression showed a significant decreasing trend from distant normal stomach mucosa to adjacent non-tumor tissues and further to primary tumors (P < 0.05). Conversely, promoter methylation levels exhibited a significant increasing trend (P < 0.0001). Additionally, there was a significant inverse correlation between psiTPTE22-HERV expression and promoter methylation in these samples (r = −0.276; P = 0.0328; Fig. 2E). These findings indicate that aberrant methylation at specific CpG sites in the psiTPTE22-HERV promoter region contributes to its down-regulation in gastric cancer.

Figure 2: Regulation of psiTPTE22-HERV expression by promoter DNA methylation. (A) Silencing of psiTPTE22-HERV in 9/10 gastric cancer cell lines. (B) Restoration of psiTPTE22-HERV expression by 5-Aza demethylation treatment in gastric cancer cell lines. (C) Bisulfite genomic sequencing revealed hypermethylation of psiTPTE22-HERV promoter (11 CpG sites, underlined) in silenced gastric cancer cells, with reduced methylation in MKN1 and 5-Aza treated cells. (D) Down-regulation of psiTPTE22-HERV in primary gastric tumors versus paired normal tissues by PCR (D1; in-house samples) and RNA sequencing (D2; TCGA). (E) Progressive decrease in expression and increase in promoter methylation, observed from normal stomach mucosa to adjacent non-tumor tissues to primary tumors, along with a significant inverse correlation between expression and promoter methylation in all samples. The data were presented as mean ± standard deviation (B), mean (C), or median (interquartile range) (E) and compared using an unpaired t-test (B, C) or one-way ANOVA (E). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

psiTPTE22-HERV plays a tumor-suppressive role in vitro

To investigate its function, psiTPTE22-HERV was overexpressed in two cancer cell lines (MKN45 and SGC7901) with low endogenous expression, as confirmed by reverse transcription PCR and western blotting (Fig. 3A). Overexpression of psiTPTE22-HERV significantly inhibited cell growth and colony formation (Fig. 3B and C). Flow cytometry analysis revealed that psiTPTE22-HERV expression induced G1–S phase cell cycle arrest, with an increase in the number of G1-phase cells and a concomitant decrease in the number of S-phase cells (Fig. 3D1). This arrest was further supported by the up-regulation of G1 cell cycle inhibitors (p21 and p27) and the down-regulation of key G1 regulators (CDK4 and cyclin D3). The decrease in S-phase cells was confirmed by the down-regulated proliferation marker, proliferating cell nuclear antigen (PCNA) (Fig. 3D2). Furthermore, psiTPTE22-HERV expression significantly induced apoptosis (Fig. 3E1), accompanied by activation of caspase-9, caspase-7, caspase-3, and poly(ADP-ribose) polymerase (PARP) (Fig. 3E2). Additionally, psiTPTE22-HERV markedly decreased cell migration and invasiveness, as demonstrated by the wound healing and Matrigel invasion assays (Fig. 3F1). These findings were supported by changes in epithelial–mesenchymal transition markers, including up-regulation of the epithelial marker E-cadherin and down-regulation of mesenchymal markers, including N-cadherin, β-catenin, Vimentin, Slug, Snail, and transcription factor 8 (TCF8)/zinc finger E-box binding homeobox 1 (ZEB1) (Fig. 3F2).

Figure 3: psiTPTE22-HERV exerts tumor-suppressive effects in vitro. (A) Ectopic expression of psiTPTE22-HERV occurred in MKN45 and SGC7901 cells, as confirmed by PCR and western blotting. (B) Overexpression of psiTPTE22-HERV significantly inhibited cell viability of cancer cells. (C) Overexpression of psiTPTE22-HERV significantly inhibited colony formation of cancer cells. (D) Overexpression of psiTPTE22-HERV caused G1–S cell cycle arrest as indicated by flow cytometry. (E) Overexpression of psiTPTE22-HERV altered the expression of cell cycle-related proteins as revealed by western blotting. (F) Overexpression of psiTPTE22-HERV induced apoptosis/necrosis of cancer cells as indicated by flow cytometry. (G) Overexpression of psiTPTE22-HERV induced activation of apoptotic proteins. (H) Overexpression of psiTPTE22-HERV suppressed cell migration and invasion, as shown by wound healing and Matrigel invasion assays, respectively. (I) Overexpression of psiTPTE22-HERV altered the expression of epithelial–mesenchymal transition markers. The data were presented as mean ± standard deviation (B–F) and compared using two-way ANOVA (B) or unpaired t-test (C–F). ∗P < 0.05 and ∗∗P < 0.001.

Knockdown of psiTPTE22-HERV mitigates its tumor-suppressive role in vitro

psiTPTE22-HERV was knocked down in MKN1 cells, which exhibit high endogenous expression (Fig. 4A). psiTPTE22-HERV knockdown significantly increased cell growth (Fig. 4B) and promoted cell cycle progression, leading to reduced cells in the G1 phase and increased cells in the S and G2 phases (Fig. 4C1). These changes were accompanied by down-regulated G1 cell cycle inhibitors (p21 and p27), elevated G1 regulators (CDK4 and cyclin D3), and increased PCNA (Fig. 4C2). Furthermore, psiTPTE22-HERV knockdown significantly reduced cell apoptosis (Fig. 4D1), as evidenced by decreased activation of caspase-9, caspase-7, caspase-3, and PARP (Fig. 4D2). In addition, psiTPTE22-HERV knockdown enhanced the migration and invasion capabilities of MKN1 cells (Fig. 4E1), along with a marked decrease in E-cadherin expression and increased expression of N-cadherin, β-catenin, Vimentin, Slug, Snail, and TCF8/ZEB1 (Fig. 4E2). These findings further underscore the tumor-suppressive role of psiTPTE22-HERV.

Figure 4: Functional consequences of psiTPTE22-HERV knockdown in vitro. (A) Successful siRNA-mediated knockdown in MKN1 cells confirmed by PCR. (B) Enhanced cell viability following psiTPTE22-HERV depletion. (C) Accelerated cell cycle progression (C1) and altered cell cycle-related protein expression (C2). (D) Reduced apoptosis (D1) with decreased apoptotic protein activation (D2). (E) Increased cell migration and invasion (E1) and altered expression of epithelial–mesenchymal transition markers (E2). The data were presented as mean ± standard deviation (B–E) and compared using two-way ANOVA (B) or unpaired t-test (C–E). ∗P < 0.05, ∗∗P < 0.001, and ∗∗∗P < 0.0001.

psiTPTE22-HERV attenuates subcutaneous tumor growth and reduces metastasis of cancer cells in nude mice

To evaluate the effect of psiTPTE22-HERV on gastric tumor growth in vivo, subcutaneous xenograft tumor models were established by injecting MKN45 cells stably transfected with a psiTPTE22-HERV expression vector or an empty vector into nude mice. Tumor growth was significantly suppressed in the psiTPTE22-HERV-transfected group (Fig. 5A1). At the end of the experiment, the net weight of tumors in the psiTPTE22-HERV-transfected group was also significantly reduced (Fig. 5A2). Immunohistochemistry confirmed psiTPTE22-HERV expression in xenograft tumors in the psiTPTE22-HERV-transfected group (Fig. 5B). Consistent with the in vitro findings, psiTPTE22-HERV-expressing tumors exhibited fewer proliferating cells and more apoptotic cells, as demonstrated by the Ki-67 and TUNEL assays, respectively (Fig. 5C and D).

Figure 5: psiTPTE22-HERV suppressed subcutaneous tumor growth and metastasis in vivo. (A) Reduced subcutaneous tumor growth in nude mice with psiTPTE22-HERV-transfected cells versus controls in terms of tumor volume (A1) and tumor weight (A2). (B)psiTPTE22-HERV expression in xenografts confirmed by immunostaining. (C) Decreased proliferation in psiTPTE22-HERV-expressing tumors indicated by Ki-67 staining. (D) Increased apoptosis in psiTPTE22-HERV-expressing tumors revealed by TUNEL staining. (E) Reduced liver and lung metastasis shown by representative macroscopic images (E1) and hematoxylin-eosin staining (E2). The data were presented as mean ± standard error of the mean (A1, A2, E2) or mean ± standard deviation (B–D) and compared using two-way ANOVA (A1), unpaired t-test (A2, B–D), chi-square test, or Mann–Whitney U test (E2).

To assess the effect of psiTPTE22-HERV on tumor metastasis in vivo, SGC7901 cells stably transfected with psiTPTE22-HERV expression vector or empty vector were injected into the tail veins of female nude mice. After four weeks, metastasis to distant organs was evaluated. Histological examination revealed that 60% (6/10) of the control mice developed liver or lung metastases, compared with only 18.2% (2/11) of the mice bearing psiTPTE22-HERV-expressing cells (Fig. 5E1). The control group also exhibited significantly more metastatic lesions than the psiTPTE22-HERV group (Fig. 5E2). These results demonstrate the critical role of psiTPTE22-HERV in the suppression of cancer metastasis.

psiTPTE22-HERV disrupts protein metabolism and mTOR signaling

To elucidate the molecular mechanism of psiTPTE22-HERV, we investigated the gene expression profiles using RNA sequencing. Interestingly, the majority of the top genes down-regulated by psiTPTE22-HERV (|fold change| ≥ 2 and FDR < 0.05) were involved in protein synthesis and PI3K/AKT/mTOR signaling, including EIF4EBP1 (eukaryotic translation initiation factor 4E binding protein 1) (Fig. 6A). GO analysis of 60 differentially expressed genes (|fold change| ≥ 1.5, FDR < 0.05) revealed 10 significantly enriched biological processes (FDR < 0.05; ≥ 5 genes involved), 7 of which were associated with protein synthesis/metabolism (Fig. 6B; Tables S4 and S5). Further pathway enrichment analysis identified mTOR signaling to be dysregulated by psiTPTE22-HERV, involving the down-regulation of EIF4EBP1 (Fig. 6C; Tables S6 and S7). EIF4EBP1 plays an oncogenic role and is a key effector of PI3K/AKT/mTOR signaling.18, 19, 20 Additionally, EIF4E, a direct target of EIF4EBP1 with oncogenic roles linked to AKT and mTOR signaling,21,22 was down-regulated by psiTPTE22-HERV (fold change = −1.34).

Figure 6: Molecular mechanisms of psiTPTE22-HERV-mediated tumor suppression. (A) Heatmap of differentially expressed genes (DEGs; |fold change| ≥ 1.5 and FDR <0.05) identified by RNA sequencing, with functional annotations for top hits (|fold change| ≥ 2). (B) GO biological processes altered by psiTPTE22-HERV overexpression as identified by over-representation analysis. (C) KEGG pathways enriched by DEGs as identified by GSEA. (D) Western blotting analysis of PI3K/AKT/mTOR pathway components (PI3K, AKT, p-AKT, and mTOR), EIF4EBP1, p-EIF4EBP1(Thr70), and EIF4E. (E) Pearson's correlations of psiTPTE22-HERV expression with EIF4EBP1/EIF4E mRNA, EIF4EBP1/EIF4E protein, and p-EIF4EBP1(Thr70) protein levels in gastric tumors of the TCGA cohort. (F) Cytoplasmic-specific reduction of EIF4E by psiTPTE22-HERV, as evidenced by western blotting of cytoplasmic and nuclear fractions. (G) Immunofluorescence staining confirmation of EIF4E modulation by psiTPTE22-HERV.

psiTPTE22-HERV inhibits the PI3K/AKT/mTOR/EIF4E signaling pathway

We examined the impact of psiTPTE22-HERV on key proteins in the PI3K/AKT/mTOR signaling pathway, as well as on EIF4EBP1 and EIF4E at the protein level. Western blotting revealed that psiTPTE22-HERV down-regulated the levels of PI3K, p-AKT, mTOR, EIF4EBP1, p-EIF4EBP1 (at Thr70, driven by mTOR), and EIF4E, whereas psiTPTE22-HERV knockdown exhibited the opposite effects (Fig. 6D). Importantly, we observed significant inverse correlations between psiTPTE22-HERV expression and both EIF4E mRNA (P < 0.05) and protein levels (P < 0.0001) in primary gastric tumor tissues from the TCGA cohort (n = 331). Additionally, inverse correlations were observed with EIF4EBP1 protein (P < 0.0001) and pEIF4EBP1(Thr70) levels (P = 0.0003; Fig. 6E). Given the distinct roles of cytoplasmic and nuclear EIF4E,23 we assessed their levels in cytoplasmic and nuclear fractions. psiTPTE22-HERV down-regulated the cytoplasmic level of EIF4E, whereas psiTPTE22-HERV knockdown led to an increase in cytoplasmic EIF4E, with no changes observed in nuclear EIF4E (Fig. 6F). Further immunofluorescence staining confirmed the down-regulation of cytoplasmic EIF4E by psiTPTE22-HERV (Fig. 6G). These findings imply that the tumor-suppressive effect of psiTPTE22-HERV may be largely attributable to its negative regulation of cytoplasmic EIF4E via the PI3K/AKT/mTOR signaling pathway.

Reduced psiTPTE22-HERV expression correlates with tumor progression and poor prognosis in gastric cancer

To investigate the clinical significance of psiTPTE22-HERV, we examined its expression in a cohort of 85 Chinese gastric cancer patients from a single center. psiTPTE22-HERV expression was significantly lower in tumors than in adjacent non-tumor tissues (Fig. 7A). The expression fold change in tumor versus adjacent non-tumor tissues was assessed, revealing no correlation with clinicopathological features such as age, sex, histological type, differentiation, or lesion location (all P > 0.05; Fig. 7B). However, we observed a significant linear decrease during tumor–node–metastasis (TNM) stage progression (P < 0.0001; Fig. 7C), with notable decreases associated with T, N, and M progression (all P < 0.05; Fig. 7D). These findings align with the in vitro and in vivo findings regarding the role of psiTPTE22-HERV in cell migration and metastasis to distant organs. Univariate Cox regression analysis indicated that psiTPTE22-HERV expression and TNM stage IV/metastasis, but not other clinicopathological features (age, sex, differentiation, tumor location, Lauren type), were significantly associated with shortened survival of gastric cancer patients. Furthermore, multivariate Cox regression analysis demonstrated that psiTPTE22-HERV expression was an independent predictor of poor survival (P < 0.05; Fig. 7E). Kaplan–Meier analysis showed that low levels of psiTPTE22-HERV expression were significantly associated with shortened survival in gastric cancer patients (Fig. 7F).

Figure 7: Clinical significance of psiTPTE22-HERV in gastric cancer. (A) Significant down-regulation of psiTPTE22-HERV in tumors versus paired non-tumor tissues (85 Chinese patients). (B) No association of psiTPTE22-HERV expression (log2 of fold change (FC) in tumor versus paired non-tumor) with age, sex, histological type (1: adenocarcinoma; 2: signet ring cell carcinoma), differentiation, or tumor location. (C, D) Progressive decrease of psiTPTE22-HERV expression with TNM stage advancement and individual T/N/M category progression. (E) Poor prognostic indicators revealed by univariate and multivariate Cox analysis. (F) The Kaplan–Meier survival curve showed shortened survival in patients with low psiTPTE22-HERV expression. The data were presented as median (interquartile range) (A) or box-and-whiskers (Tukey) (B–D) and compared using Mann–Whitney U test (A, B, D-right) or one-way ANOVA (C, D).