Inhibition of the PI3K/AKT signaling pathway blocks the oncogenic activity of TRIM26 in prostate cancer cells

Abstract

The tripartite motif-containing protein 26 (TRIM26) is an E3 ubiquitin ligase with strong oncogenic activity in various cancers. However, its role and molecular mechanism in prostate cancer (PCa) remain elusive. To clarify its role in promoting PCa progression, we evaluated TRIM26 expression in cells and clinical specimens using immunohistochemistry and found that TRIM26 was significantly upregulated in PCa tissue. Moreover, high TRIM26 levels predicted a poor prognosis in patients with PCa. Ectopic overexpression of TRIM26 increased PCa cell proliferation and migration, and this activity was suppressed by TRIM26 knockdown. Notably, TRIM26 activated both protein kinase B (AKT)/mammalian target of the rapamycin (mTOR) and the epithelial–mesenchymal transition (EMT) signaling pathways in PCa cells. Consistent with these findings, TRIM26 knockdown led to decreased activation of these signals. Furthermore, the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway activated by TRIM26 was attenuated by the PI3K inhibitor S14161. Similarly, cisplatin, a commonly used anti-PCa drug, downregulated TRIM26 and AKT/mTOR activation, while TRIM26 overexpression reversed AKT/mTOR inactivation. Finally, this finding was also demonstrated TRIM26 expression strikingly promoted tumor growth and activated AKT/mTOR signaling in a PCa xenograft. In conclusion, TRIM26 drives PCa malignancy and may be an attractive target for PCa treatment.

INTRODUCTION

Prostate cancer (PCa) is a very common malignancy in men. In the USA, it has been estimated that over 299 010 new PCa cases were diagnosed and approximately 35 250 men died from PCa in 2024. Over the past decade, the proportion of patients diagnosed with metastatic PCa increased from 3.9% to 8.2%.1 Early diagnosis is inadequate because PCa may have no signs or symptoms in its early stages. When diagnosed, most patients have advanced disease. The treatment modalities for metastatic PCa mainly include androgen deprivation therapy and chemotherapy.2 However, resistance and relapse are common even after intensive treatment. Identification of new drugs and targets to treat PCa are urgently needed.

The tripartite motif-containing protein 26 (TRIM26) is a member of the TRIM proteins family composed of multidomain E3 ubiquitin ligases.3 Current studies report that TRIM26 is mainly involved in viral replication and cancer cell proliferation.4 As a ubiquitin ligase, TRIM26 mediates and promotes hepatitis B x (HBx) and nucleocapsid protein degradation, thereby preventing hepatitis B virus (HBV) replication and porcine reproductive and respiratory viral infection.5 In addition, TRIM26 displays strong oncogenic activity in various cancers, including hepatocellular carcinoma (HCC),6 bladder cancer,7 glioblastoma,8 nasopharyngeal carcinoma,9 papillary thyroid carcinoma,10 and non-small cell lung cancer (NSCLC).11 However, the expression of TRIM26 and its activities in PCa are not yet known.

MATERIALS AND METHODS

Cell culture and chemicals

PCa cell lines (PC-3, DU145, and RM-1) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in RPMI-1640 medium. Human prostatic stromal immortalized cells (WPMY-1) and human embryonic kidney cells (HEK293T) were cultured with Dulbecco’s high-glucose modified Eagle’s medium (Thermo Fisher Scientific, Shanghai, China). All media were supplemented with 100 units per ml of streptomycin, 100 μg ml⁻¹ of penicillin, and 10% fetal bovine serum (Vazyme, Nanjing, China). S14161 and cisplatin were purchased from Maybridge Chemical Co., Ltd. (Tintagel, UK) and Selleck Chemicals (Shanghai, China), respectively.

PCa tissues

After obtaining approval from the Review Board of Medical Ethics of Jinling Hospital (Nanjing, China; Approval No. 2022NZKY-049-02) and written consent from patients, clinical tissue samples were collected from 45 patients undergoing prostatectomy, transurethral resection of the prostate, or biopsy. There were 9 cases of benign prostate hyperplasia (BPH), 9 cases of prostatic intraepithelial neoplasia (PIN), and 27 cases of PCa.

TRIM26 plasmid and transfection

Polymerase chain reaction (PCR) was used to clone the TRIM26 gene. The specific primers were as follows: forward, 5’-GCGCGGATCCATGGCCACGTCAGCCCCACTAC-3’; and reverse, 5’-GCGOGGGCCCGAGGGTCTTAGCAGGAGGCGTGTTC-3’. Then, the PCR product was inserted into a pcDNA3.1 vector. The TRIM26 expression plasmids were transfected into PCa cells according to the manufacturer’s protocol. To maintain the transfection efficiency at about 70%–80%, the appropriate number of plasmids was determined when cells were split for further studies.

Small interfering RNA (siRNA)

The specific siRNAs of TRIM26 were reported in our recent study.11 These siRNAs were synthesized by RiboBio (Guangzhou, China) and were transfected into PCa cells using Lipofectamine 2000® reagent (Invitrogen, Calsbard, CA, USA) as the gene carrier.

Western blot (WB) analysis

Cell lysates were prepared, and protein concentrations were quantified using bicinchoninic acid assays. Equal amounts (40 μg) of total proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and subsequently incubated with the following antibodies: monoclonal anti-Myc (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan); antibodies against TRIM26, p53, p21, phosphatase and tensin homolog deleted on chromosome ten (PTEN), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), all from Proteintech, Wuhan, China; and primary antibodies against E-cadherin, N-cadherin, phosphorylated protein kinase B (p-AKT), AKT, phosphorylated mammalian target of the rapamycin (p-mTOR), and mTOR (Cell Signaling Technology, Inc., Danvers, MA, USA). Then, the protein concentrations were analyzed.

5-ethynyl-2’-deoxyuridine (EdU) incorporation assay

To assess cell proliferation, EdU incorporation assays were performed using Apollo® fluorescent dyes (Cell-Light™ EdU Apollo567 In Vitro Kit; RiboBio) according to the manufacturer’s instructions. Finally, the cells were analyzed using a fluorescence microscope (Carl Zeiss Industrielle Messtechnik GmbH, Oberkochen, Germany).12

Transwell migration assays

Transwell assays were performed as described in detail previously.12 After staining with 1% crystal violet solution, the migrated cells were analyzed using a light microscope (Nikon, Tokyo, Japan), and counted using Image J software (National Institutes of Health, Baltimore, MD, USA).

Immunohistochemistry (IHC)

Analysis of TRIM26 expression in primary PCa and associated tissues was performed by IHC as described in detail previously.13 Specifically, the tissues of interest were embedded in paraffin, and 5-μm longitudinal sections were prepared with a Leica rotary microtome (Wetzlar, Germany) and mounted on slides. The slides were deparaffinized with xylene, and then rehydrated with ethanol and citric acid solution. The slides were then incubated stepwise with 3% hydrogen peroxide solution, 3% bovine serum albumin (BSA)-containing blocking solution, and an anti-TRIM26 antibody. Finally, the sections were counterstained with hematoxylin. Staining intensity was scored according to a semiquantitative four-grade scale that was described previously.13

Murine PCa model

A murine PCa cell line (RM1) was stably transfected with Myc-TRIM26 or empty vector. A total of 5 × 105 cells per site were subcutaneously injected into the flank of male BALB/c mice from Guangdong Vital River Laboratory Animal Technology Co., Ltd. (Foshan, China). Tumor growth and body weight were monitored daily for 1 week. At the end of the study, tumors were removed, weighed, and subjected to Western blot to examine AKT/mTOR signaling protein expression. This animal study was approved by the Institutional Animal Care and Use Committee of Guangdong Zhiyuan Biomedical Technology Co., Ltd. (Guangzhou, China; Approval No. IAEC-2024121801).

Statistical analyses

All experiments were performed in triplicate, and the results are shown as mean ± standard deviation (s.d.) where applicable. Results were analyzed using Student’s t-tests or one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.

RESULTS

TRIM26 is highly expressed in PCa tissues and is associated with poor outcomes in patients with PCa

The TRIM26 oncogene has been reported in various cancers, but its pattern of expression across different cancers is not yet known. To this end, we analyzed the spectrum of TRIM26 expression in more than 20 cancers based on the Cancer Genome Atlas (TCGA) data from The Human Protein Atlas database (https://www.proteinatlas.org/), as of March 10, 2024. The results showed that TRIM26 is highly expressed in more than 90% of PCa tissues, second to that in testis cancers (Figure 1a). In contrast, there was less TRIM26 expression in lung cancer tissues (Figure 1a). This was consistent with IHC findings showing that TRIM26 was notably increased in PCa tissue compared with NSCLC tissue (Figure 1b). Typical results of IHC staining assays were shown in Figure 1c. Moreover, TRIM26 expression was negatively associated with overall survival in patients with PCa (Figure 1d). We also examined TRIM26 expression in a panel of prostate tissues from patients with BPH, PIN, or PCa. TRIM26 expression was higher in PCa tissues than in tissues from patients with BPH and PIN (Figure 1e). The TRIM26 expression rate in PCa tissues was more than 70% (Figure 1f). Therefore, TRIM26 is likely to be a novel prognostic biomarker in patients with PCa.

Figure 1.

TRIM26 is highly expressed in PCa and is associated with poor prognosis in patients with PCa. (a) Expression profile of TRIM26 in various cancer tissues assayed based on the Human Protein Atlas. (b) Comparative expression of TRIM26 in PCa and lung cancer. (c) Representative immunohistochemical staining of TRIM26 in normal prostate tissues and PCa tissues. (d) Association between TRIM26 expression and overall survival in patients with PCa (n = 246 for each group). (e) Representative immunohistochemical staining of TRIM26 in BPH, PIN, and PCa tissues. (f) Proportion of TRIM26 expression in BPH, PIN, and PCa tissues. BPH: benign prostate hyperplasia; PCa: prostate cancer; PIN: prostatic intraepithelial neoplasia; TRIM26: the tripartite motif-containing protein 26; TCGA: The Cancer Genome Atlas; HR: hazard ratio; PRAD: prostate adenocarcinoma.

TRIM26 promotes PCa cell proliferation and migration

Since TRIM26 is highly expressed in PCa tissues, which indicates a poor prognosis, we explored the effects of TRIM26 on PCa cell activity. TRIM26 was overexpressed or knocked down in PC-3 cells, and EdU corporation assays were performed. EdU is an analog of uridine and can replace the thymine deoxyriboside and be incorporated into newly synthesized DNA during cell proliferation. The results revealed a striking increase in EdU-positive cells when TRIM26 was overexpressed in PC-3 cells, whereas EdU-positive cells were significantly decreased when TRIM26 was knocked down (Figure 2a–2d). These findings suggest that TRIM26 promotes PCa cell proliferation. Given that metastasis is common in patients with PCa, we next investigated whether TRIM26 contributes to PCa metastasis by evaluating the role of TRIM26 in PCa migration. After TRIM26 was upregulated or knocked down in PC-3 cells, transwell assays were performed. The results showed that TRIM26 upregulation markedly increased cell migration and invasion of PC-3 cells; in contrast, TRIM26 knockdown markedly inhibited the migration and invasion of related cells (Figure 2e–2h). We also analyzed the effects of TRIM26 on prostate stromal cells, because these cells are critical for PCa development and invasion.14,15 Similar results were observed using WPMY-1 cells derived from the stroma of the prostate. TRIM26 increased stromal cell proliferation and migration (Figure 2a and 2e), whereas these activities were markedly reduced following TRIM26 knockdown (Figure 2b and 2f). Together, these findings suggest that TRIM26 promotes PCa cell proliferation, migration, and invasion.

Figure 2.

TRIM26 promotes PCa cell migration and proliferation. PC-3 and WPMY-1 cells were transfected with (a) Myc-TRIM26 or (b) siTRIM26, respectively, for 24 h, then EdU incorporation assays were performed. Statistical analyses based on EdU-positive cells were shown in (c) TRIM26 and (d) siTRIM26, respectively. PC-3 and WPMY-1 cells were transfected with (e) TRIM26 or (f) siTRIM26, respectively, for 24 h, then transwell migration assays were performed. Statistical analyses based on EdU-positive cells were shown in (g) TRIM26 and (h) siTRIM26, respectively. ^***P < 0.001. EdU: 5-ethynyl-2’-deoxyuridine; TRIM26: the tripartite motif-containing protein 26; si: small interfering; DAPI: 4’,6-diamidino-2-phenylindole.

TRIM26 activates the AKT/mTOR signaling pathway and epithelial–mesenchymal transition (EMT) signaling in PCa cells

The above studies indicate that TRIM26 is highly expressed in PCa and promotes the proliferation and migration of PCa cells. Since the PTEN/AKT/mTOR pathway may regulate cancer cell migration and EMT, we investigated whether TRIM26 activates AKT/mTOR and EMT signaling. TRIM26 was transfected into PCa cells. As shown in Figure 3a, the phosphorylation levels of both AKT and mTOR proteins were upregulated by TRIM26. In contrast, p-AKT and p-mTOR were markedly decreased when TRIM26 was knocked down (Figure 3b), indicating that TRIM26 activated the AKT/mTOR signaling pathway. Furthermore, we found that in both PC-3 and DU145 cells, TRIM26 overexpression inhibited E-Cadherin expression but increased the expression of N-Cadherin, a critical signal in the EMT process (Figure 3c). Notably, these changes were downregulated when TRIM26 was knocked down (Figure 3d), suggesting that TRIM26 promoted EMT in PCa cells. Given that chemotherapy is a common treatment for metastatic PCa, we also examined the effects of cisplatin on TRIM26 expression and AKT/mTOR signaling. Results showed that cisplatin downregulated TRIM26 and inhibited AKT and mTOR activation in a concentration-dependent manner (Figure 3e). Moreover, the signals inhibited by cisplatin were rescued by TRIM26 overexpression (Figure 3f). These findings suggest that TRIM26 may act mainly on cell proliferation, migration, and invasion signals via activating the AKT/mTOR signaling pathway.

Figure 3.

TRIM26 activates AKT/mTOR and EMT signaling in PCa cells. DU145 and PC-3 cells were transfected with (a) TRIM26 plasmids or (b) siTRIM26 for 24 h, then Western blot was performed to assess the AKT signaling pathway. DU145 and PC-3 cells after transfection with (c) TRIM26 plasmids or (d) its siRNA were subjected to Western blot to assess EMT signaling. (e) DU145 and PC-3 cells were treated with increased CDDP for 24 h, then cell lysates were prepared and Western blot performed as indicated. (f) DU145 and PC-3 cells were infected with TRIM26 lentivirus for 48 h, treated with CDDP for 24 h, then cell lysates were prepared and Western blot performed as indicated. AKT: protein kinase B; CDDP: cisplatin; EMT: epithelial-mesenchymal transition; TRIM26: the tripartite motif-containing protein 26; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; mTOR: mammalian target of rapamycin; p-mTOR: phosphorylated mTOR; AKT: protein kinase B; p-AKT: phosphorylated AKT.

S14161 inhibits PCa cell migration by suppressing the TRIM26/AKT/mTOR signaling pathway

S14161 is a specific inhibitor of the PI3K/AKT/mTOR signaling pathway.15 Because TRIM26 activates the AKT/mTOR signaling, we investigated whether S14161 could inhibit the migration of PCa cells by inhibiting TRIM26/AKT signaling. To verify this hypothesis, PCa cells were treated with S14161 for 24 h; subsequent Western blot indicated that S14161 markedly downregulated TRIM26 and suppressed the phosphorylation of AKT and mTOR in PCa cells in a concentration-dependent manner (Figure 4a), suggesting that S14161 suppresses the TRIM26/AKT/mTOR pathway. Moreover, transwell migration assays showed that S14161 strikingly inhibited PCa cell migration (Figure 4b and 4c). These results suggest that S14161 may inhibit the AKT/mTOR signaling by targeting TRIM26. To verify the impact of S14161 on the TRIM26/AKT signaling pathway, PCa cells were overexpressed with TRIM26, and then treated with S14161. Western blot showed that overexpression of TRIM26 abolished the inhibition of AKT and mTOR phosphorylation by S14161 (Figure 4d). Moreover, the PCa cell migration and invasion inhibited by S14161 were rescued by TRIM26 overexpression (Figure 4d–4f). Therefore, inhibiting the AKT/mTOR signaling pathway blocks TRIM26-promoted PCa cell proliferation, migration, and invasion.

Figure 4.

Inhibition of the AKT/mTOR signaling pathway abolishes TRIM26 activity and inhibits PCa cell migration. DU145 and PC-3 cells were treated with the PI3K inhibitor S14161 for 24 h, and the following procedures were performed: (a) Western blot for specific proteins as indicated; (b) transwell migration assays, and (c) statistical analysis of migrated cells. (d–f) DU145 and PC-3 cells were transfected with TRIM26 plasmids for 24 h and treated with S14161 for 24 h. Then, the following were performed: (d) Western blot for specific proteins as indicated; (e) transwell migration assays; and (f) statistical analysis of migrated cells. ^***P < 0.001. PI3K: phosphoinositide 3-kinase; TRIM26: the tripartite motif-containing protein 26; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; AKT: protein kinase B; p-AKT: phosphorylated AKT; mTOR: mammalian target of rapamycin; p-mTOR: phosphorylated mTOR.

TRIM26 promotes PCa tumor growth in vivo

The studies described above have demonstrated that TRIM26 promotes PCa cell proliferation and survival in cultured cells. To determine whether TRIM26 displayed similar activity in PCa in vivo, we established a murine PCa model using a TRIM26-overexpressing murine PCa cell line (RM-1). TRIM26 strikingly promoted PCa tumor growth in mice injected with stably transfected cells compared with control mice that received cells transfected with empty vector (Figure 5a–5c). Moreover, the analysis of tumor tissues showed that AKT/mTOR signals were markedly activated, and EMT marker proteins, including N-Cadherin and vimentin, were upregulated in tumors derived from TRIM26-overexpressing cells (Figure 5d). These results were consistent with the in vitro findings. Therefore, TRIM26 promotes PCa via activation of the AKT/mTOR signaling pathway.

Figure 5.

TRIM26 promotes PCa tumor growth in vivo in association with activation of AKT/mTOR and EMT signals. RM-1 cells were injected subcutaneously into BALB/c male mice at a density of 5 × 105 cells per site. (a) Tumor growth was monitored every day for 1 week. Tumors were (b) removed and (c) weighed. (d) Proteins were extracted from tumor tissues and Western blot was performed for specific proteins as indicated. ^***P < 0.001. AKT: protein kinase B; EMT: epithelial-mesenchymal transition; mTOR: target of rapamycin; PCa: prostate cancer; TRIM26: the tripartite motif-containing protein 26; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; pCDH-NC: pCDH plasmid-negative control; p-AKT: phosphorylated AKT; p-mTOR: phosphorylated mTOR.

DISCUSSION

Our study indicates that TRIM26 activates AKT/mTOR and EMT signaling, thereby promoting the proliferation, migration, and invasion of PCa cells. Moreover, inhibiting PI3K/AKT signaling blocks TRIM26 activity.

TRIM26 is a ubiquitin ligase that has diverse functions in different cancers. In some cancers, including HCC and thyroid cancer, TRIM26 displays suppressive activity;10,16 in contrast, it activates signals involved in cancer progression in NSCLC and glioblastoma.7,11 In this study, TRIM26 expression was very high in PCa tissues. Functionally, TRIM26 promoted PCa cell proliferation and migration. Mechanistically, TRIM26 may mainly act as a ubiquitin ligase. TRIM26 induces sex-determining region Y-box2 (SOX2) degradation in glioblastoma8 and mediates the degradation of zinc finger E-box binding homeobox 1 (ZEB1) in hepatocellular carcinoma (HCC) cells.6 Our recent research shows that TRIM26 directs pre-B-cell leukemia homeobox transcription factor 1 (PBX1) for K48-linked polyubiquitination and degradation, thereby promoting NSCLC cell proliferation and migration.11 Although we did not identify the substrate proteins of TRIM26 in PCa in the present study, we found that TRIM26 may promote PCa cell proliferation, migration, and invasion via the PI3K/AKT pathway.

The PI3K/AKT/mTOR signaling pathway is the central node of signaling hubs that control a wide variety of cellular events, including cell proliferation and survival via downstream signaling cascades.17 Notably, castration-resistant PCa typically involves PI3K/AKT/mTOR pathway abnormalities.18 Our previous study demonstrated that overactivation of the PI3K/AKT/mTOR pathway contributes to PCa chemoresistance and that inhibiting PI3K/AKT can induce PCa cell apoptosis.19 Recent research suggests that PI3K/AKT overactivation confers a more progressive phenotype in PCa cells.20 Therefore, the PI3K/AKT pathway is believed to be an ideal therapeutic target in PCa. However, PI3K/AKT can be activated by diverse extracellular signals as well as endogenous molecules including, but not limited to, receptor tyrosine kinases, non-receptor tyrosine kinases, and G-protein coupled receptor signals.21 This study shows that TRIM26 is also an important regulator in the PI3K/AKT/mTOR signaling pathway. Although PTEN is a braker protein that inhibits the PI3K/AKT signaling pathway, it was not altered by TRIM26 (Figure 3d). Interestingly, other major inhibitors of carcinogenesis and cancer progression, including p53 and p21, were not downregulated by TRIM26 either. In contrast, TRIM26 strikingly activated both AKT and mTOR via phosphorylation. In addition, TRIM26 also upregulated the expression of N-Cadherin and downregulated the expression of E-Cadherin, two hallmarks of EMT in cancer cells; these changes promote both metastasis and drug resistance. Notably, AKT/mTOR signals are the major drivers of EMT.22 Given that EMT is a precondition of cell migration, these findings further demonstrate that targeting the TRIM26/AKT/mTOR signaling axis could help prevent the proliferation, migration, and invasion of PCa cells. However, the mechanism by which TRIM26 activates the AKT/mTOR pathway requires further study.

In conclusion, TRIM26 is highly expressed in PCa and serves as an oncogene by activating AKT/mTOR and promoting EMT. We also demonstrated that inhibiting the PI3K/AKT/mTOR pathway abolishes TRIM26 activity in PCa. The TRIM26/AKT/mTOR axis could be a novel target for PCa treatment.

AUTHOR CONTRIBUTIONS

YYZ, LHZ, XYZ, XMZ, and YMH performed the experiments. YYZ, XLM, and XXF interpreted the data and prepared the manuscript. XMZ and XFX performed the patients’ studies. XLM and XFX designed the experiments and finalized the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.