PACT Inhibits Malignant Biological Behavior of Ovarian Cancer Cells by Regulating TERT Expression

ABSTRACT

Ovarian cancer (OC) is a lethal malignancy with limited treatment options. While p53‐associated cellular protein‐testes derived (PACT) has been implicated in cellular stress responses, its role in OC progression and the regulation of ferroptosis remains unclear. The functional role of PACT was examined in A2780 and OVCAR‐3 cells through siRNA‐mediated knockdown and overexpression. The CCK‐8 assay was used to evaluate cell proliferation, and the transwell assay was used to detect cell migration and invasion. Ferroptosis was assessed by measuring cell death, iron content, malondialdehyde (MDA), reactive oxygen species (ROS), and the expression of key proteins (SLC7A11, GPX4). Molecular mechanisms were investigated via co‐immunoprecipitation (Co‐IP), ubiquitination assays, and analysis of subcellular localization. In vivo validation was conducted using a xenograft model in BALB/c nude mice. The results showed that PACT knockdown significantly inhibited the proliferation, migration, and invasion of OC cells. It also promoted ferroptosis, as indicated by elevated iron accumulation, lipid peroxidation, and ROS levels, along with reduced SLC7A11 and GPX4 expression—effects that were reversed by ferrostatin‐1. PACT was found to enhance TERT expression and telomerase activity via the Keap1‐Nrf2 pathway by facilitating Keap1 ubiquitination and degradation, which promoted Nrf2 nuclear translocation. Importantly, Nrf2 knockdown abolished PACT‐mediated TERT upregulation. In vivo, PACT silencing suppressed tumor growth and induced ferroptosis. In conclusion, PACT promotes OC progression by suppressing ferroptosis and enhancing malignant phenotypes through the Keap1‐Nrf2‐TERT axis. These findings suggest that targeting PACT may represent a promising therapeutic strategy for OC.

1. Introduction

Ovarian cancer (OC) is one of the common and deadly malignant tumors in the female reproductive system [1, 2]. Although the incidence rate of OC is lower than that of cervical cancer and endometrial cancer, ranking third in gynecological malignancies, the mortality rate of OC is higher than that of cervical cancer and endometrial cancer combined, and it is the biggest disease that seriously threatens women's health [3]. The early symptoms of OC are not obvious, and most patients are already in the late stage when diagnosed, which poses a huge challenge to treatment [4]. At present, the main treatment methods for OC include surgical resection, chemotherapy, and radiotherapy, but the treatment effect is limited, and the prognosis of patients is poor [5, 6]. Therefore, it is particularly important to explore the pathogenesis of OC and search for new therapeutic targets.

Telomerase is an enzyme responsible for the elongation of telomeres in cells [7, 8]. And it is a basic nuclear protein reverse transcriptase that can add telomeric DNA to the ends of eukaryotic cell chromosomes, fill in the telomeres lost during DNA replication, repair, and elongate telomeres, prevent them from being damaged by cell division, and increase the number of cell divisions [9, 10]. Telomerase activity is inhibited in most normal tissues, but widely activated in tumor cells [11, 12]. Telomerase is mainly composed of telomerase RNA template, telomerase reverse transcriptase (TERT), and telomerase‐related proteins [13]. TERT, as a catalytic subunit of telomerase, synthesizes telomere DNA through reverse transcription to maintain telomere length and ensure the potential for sustained cell division [14]. The higher the expression level of TERT, the stronger the activity of telomerase. Inhibition of TERT reduces telomerase activity, leading to the death of OC cells [15].

The p53‐associated cellular protein‐testes derived (PACT), also known as RBBP6, P2P‐R, PP‐RP or RBQ‐1, is a nuclear protein containing a conserved N‐terminal ring finger domain, which has been reported to be involved in regulating protein SUMOylation and ubiquitination [16]. Research reports that PACT is abnormally expressed in various cancers and is closely related to cancer progression [17]. A cervical cancer study found that knocking out PACT resulted in reduced TERT expression and decreased telomerase activity [18]. It is speculated that PACT may participate in cancer progression by regulating TERT and telomerase activity. However, the role of PACT in OC has not been fully elucidated, especially its role in ferroptosis in OC.

The main objective of this study is to investigate the role of PACT in the malignant phenotype and ferroptosis of OC, and to explore the possible mechanism of action based on TERT. This study found that PACT is involved in the proliferation, migration, invasion, and ferroptosis of OC cells by regulating the expression of TERT. The above results indicate that PACT may be a potential target for treating OC.

2. Materials and Methods

2.1. Human Tissue Samples

A total of 60 pairs of human ovarian cancer tissues and matched adjacent normal ovarian tissues were obtained from patients who underwent surgical resection at the First Affiliated Hospital of Xi'an Jiaotong University. All patients were histopathologically diagnosed with ovarian cancer, and none had received preoperative radiotherapy, chemotherapy, or other neoadjuvant therapies. Fresh tissue samples were collected immediately after resection, snap‐frozen in liquid nitrogen, and stored at −80°C for subsequent protein and RNA extraction. For immunohistochemical analysis, additional tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. Written informed consent was obtained from all patients, and the study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University.

2.2. Animal Model

BALB/c male nude mice were kept in an environment with a temperature of (22 ± 1°C) and humidity of (55 ± 5%), with a light/dark cycle of 12 h, and had free access to water and food. For in vivo studies, A2780 cells were transduced with recombinant adenovirus carrying siRNA targeting PACT (Ad‐si‐PACT) or negative control siRNA (Ad‐si‐NC) at a multiplicity of infection of 50 for 48 h. After confirming efficient knockdown by Western blot, cells were harvested and subcutaneously injected into BALB/c nude mice. Mice were randomly divided into two groups: control si‐NC and si‐PACT (n = 5 per group). Tumor volume and weight were measured as described. After 4 weeks of injection, the mice were euthanized, and the tumors were collected for photography and weighing. The animal experiment has been approved by the Ethics Committee of The First Affiliated Hospital of Xi'an Jiaotong University.

2.3. Cell Culture and Treatment

Human OC cell lines A2780 and OVCAR‐3 were cultured in RPMI‐1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a 5% CO₂ atmosphere. Cells were transfected with siRNA or overexpression plasmids using Lipofectamine 3000. For functional assays, cells were treated with Ferrostatin‐1 (1 μM), MG132 (10 μM), or cycloheximide (CHX, 50 μg/mL) at indicated concentrations and durations.

2.4. Transfection

The pcDNA3.1‐TERT and siRNA of PACT (si‐PACT) were transfected into cells with Lipo2000. Forty‐eight hours later, lentivirus was harvested and used to infect OC cells.

2.5. CCK‐8 Assay

The cells were inoculated into 96‐well plates, and then the appropriate amount of CCK‐8 reagent (Dojindo Chemical Laboratory, Kumamoto, Japan) was added to each well. The plates were incubated in an incubator to fully react the cells with the CCK‐8 reagent. The OD values were measured at 450 nm every 24 h until 96 h.

2.6. MDA and Total Iron Content

The MDA assay kits and the total iron content assay kits (Abcam, MA, USA) were used to measure the MDA and total iron contents, respectively, according to the instructions.

2.7. Cell Migration

Cells were cultured to the logarithmic growth phase, digested with trypsin, centrifuged, and resuspended in serum‐free RPMI‐1640 medium. A total of 1 × 10⁵ cells in 200 μL of serum‐free medium were added to the upper chamber of a Transwell insert (Corning, NY, USA) with 8‐μm pore size. The lower chamber was filled with 600 μL of RPMI‐1640 medium containing 10% fetal bovine serum as a chemoattractant. After incubation for 24 h at 37°C in a 5% CO₂ atmosphere, the non‐migrating cells on the upper surface of the membrane were gently removed with a cotton swab. Cells that had migrated to the lower surface were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet (Merck, MA, USA) for 20 min at room temperature. The stained cells were observed and counted in five randomly selected fields per well under an inverted microscope (Olympus Corporation, Tokyo, Japan).

2.8. Cell Invasion

Cells were cultured to the logarithmic growth phase, digested, centrifuged, and resuspended in a serum‐free medium. The appropriate amount of cell suspension was added to the upper chamber of the transwell, and serum‐containing medium was added to the lower chamber. Twenty‐four hours later, the transwell was removed, and cells were fixed and stained using crystal violet (Merck, MA, USA). The cells that migrated to the lower chamber were observed and counted under the microscope (Thermo Fisher Scientific, MA, USA).

2.9. ROS Levels

DCFH‐DA was diluted with serum‐free culture medium at a ratio of 1:1000. After cell collection, suspend in diluted DCFH‐DA and incubate in a 37°C‐cell culture incubator for 20 min. Wash the cells three times with serum‐free cell culture medium to remove DCFH‐DA that has not entered the cells. Cells were collected and detected using a flow cytometer.

2.10. Cell Death Analysis

For cell death analysis, cells were harvested after the indicated treatments and stained with Annexin V‐FITC and propidium iodide (PI) using an apoptosis detection kit according to the manufacturer's protocol. Flow cytometric analysis was performed using a flow cytometer (instrument model and manufacturer). Unstained cells, single‐stained cells (Annexin V‐FITC only and PI only), and isotype controls were used as negative controls to set the compensation and define the quadrants. Debris and cell doublets were excluded by gating on forward scatter (FSC) versus side scatter (SSC) profiles. Annexin V‐FITC fluorescence was detected in the FITC channel (excitation 488 nm, emission 525 nm), and PI fluorescence was detected in the PE channel (excitation 488 nm, emission 585 nm). A minimum of 10,000 events was acquired per sample, and data were analyzed using FlowJo software. All experiments were repeated independently at least three times, and consistent gating strategies were applied across all experimental groups.

2.11. Telomerase Activity Assay

The determination of telomerase activity was carried out according to the PCR enzyme‐linked immunosorbent assay instructions of Roche Diagnostics GmbH (Mannheim, Germany). For specific experimental details as previously described [15].

2.12. Ubiquitination Assay

Cells were transfected with indicated plasmids and treated with 10 μM MG132 for 6 h before harvesting. Lysates were immunoprecipitated with anti‐Keap1 antibody overnight at 4°C, followed by incubation with Protein A/G beads. Immunoprecipitates were analyzed by Western blot using an anti‐ubiquitin antibody. Input lysates and IgG controls were included to ensure specificity. Ubiquitination levels were quantified by measuring poly‐ubiquitinated Keap1 bands. Experiments were repeated three times independently.

2.13. Co‐Immunoprecipitation (Co‐IP)

Cells were lysed in IP buffer containing protease inhibitors. Lysates were incubated with anti‐PACT or anti‐Keap1 antibody overnight at 4°C with gentle rocking, followed by incubation with Protein A/G beads for 2 h. Beads were washed extensively with lysis buffer, and bound proteins were eluted in SDS loading buffer. Immunoprecipitates and input controls were analyzed by Western blot using corresponding antibodies. Normal IgG was used as a negative control.

2.14. RT‐qPCR

Total RNA was extracted from different cell lines, and subsequently, the cDNA was obtained using the Prime Script RT Master Mix (Takara, Kusatsu, Japan). Then, cDNA was used as a template for PCR amplification. The 2^−ΔΔCt method was applied to evaluate the relative mRNA expression of PACT.

2.15. Western Blot

Total cellular proteins were extracted, and protein concentrations were determined using the BCA Protein Assay Kit (Qiagen, NRW, Germany). Each protein samples were subjected to 10% SDS‐PAGE, transferred to PVDF membranes (Millipore, MA, USA) and incubated overnight at 4°C with primary antibodies against PACT (1:1000), TERT (1:000), SLC7A11 (1:1000), GPX4 (1:1000), Keap1 (1:1000), Nrf2 (1:1000) or GAPDH (1:2000). After washing with TBST solution (Takara, Shiga, Japan), the membranes were incubated with secondary antibody (1:5000) for 1 h. The signal was shown by the enhanced chemiluminescence immunoblotting detection reagent (Thermo Fisher Scientific, MA, USA).

2.16. Statistical Analysis

To ensure the reliability of the results, all experiments were replicated across a minimum of three distinct groups, and the findings were expressed as “mean ± SEM”. When comparing just two groups, we relied on the t‐test. However, for assessing disparities among multiple groups, we adopted one‐way ANOVA followed by Tukey's post hoc test. Statistical significance was defined as p < 0.05.

3. Results

3.1. PACT Was Upregulated in Human Ovarian Cancer Tissues and Cell Lines

The expression of PACT was evaluated in clinical specimens and ovarian cancer cell lines. As shown in Figure 1A, the relative PACT mRNA level was significantly higher in 60 human ovarian cancer tissues compared with 60 adjacent normal tissues. Western blot analysis of six paired normal (N) and tumor (T) tissues further confirmed that PACT protein expression was markedly elevated in tumor samples (Figure 1B). Immunohistochemical staining (Figure 1C) also revealed stronger PACT signals in ovarian cancer tissues than in normal controls. To extend these observations to cell line models, PACT mRNA levels were examined in a panel of human ovarian cancer cell lines (A2780, HEY, RMG‐1, CAOV3, OVCA420 OVCAR‐3, SK‐OV‐3) and compared with human ovarian surface epithelial cells (HOSEpic). All cancer cell lines showed substantially higher PACT mRNA expression than HOSEpic cells (Figure 1D). Consistently, Western blot analysis confirmed elevated PACT protein expression in the same panel of ovarian cancer cell lines (Figure 1E). Collectively, these data demonstrate that PACT was upregulated both in human ovarian cancer tissues and in multiple ovarian cancer cell lines, suggesting a potential oncogenic role in ovarian cancer.

Figure 1.

PACT was upregulated in human ovarian cancer tissues and cell lines. (A) Relative PACT mRNA levels in 60 human ovarian cancer tissues compared with 60 adjacent normal tissues, as determined by RT‐qPCR. (B) Western blot analysis of PACT protein expression in six paired normal (N) and tumor (T) ovarian cancer tissues. (C) Immunohistochemical staining of PACT in representative normal and ovarian cancer tissue samples. (D) Relative PACT mRNA levels in human ovarian surface epithelial cells (HOSEpic) and a panel of ovarian cancer cell lines (A2780, HEY, RMG‐1, CAOV3, OVCA420 OVCAR‐3, SK‐OV‐3). (E) Western blot analysis of PACT protein expression in the same panel of cell lines. *p < 0.05 vs. normal tissues or vs. HOSEpic cells.

3.2. Knockdown of PACT Inhibited Proliferation, Migration, and Invasion of OC Cells

RT‐qPCR was performed using two pairs of PACT‐specific primers (PACT‐1 and PACT‐2). The results showed that all three siRNAs effectively knocked down PACT expression, with knockdown efficiencies exceeding 70%. Among them, si‐PACT #1 displayed the highest interference efficiency and was therefore selected for further experiments (Figure 2A–D). CCK‐8 assays revealed that PACT knockdown significantly suppressed the proliferation of A2780 and OVCAR‐3 cells (Figure 2E,F). The BrdU assay further revealed a significant decrease in cell proliferation ability after PACT silencing (Figure 2G,H). The results indicated that reduced PACT expression impaired cell migration in both cell lines (Figure 2J,K). Furthermore, Transwell invasion assays demonstrated that PACT interference markedly inhibited the invasive ability of A2780 and OVCAR‐3 cells (Figure 2L,M). Collectively, these findings suggest that silencing PACT restrains the proliferative, migratory, and invasive capacities of ovarian cancer cells.

Figure 2.

Interference with PACT inhibited proliferation, migration, and invasion of OC cells. A2780 and OVCAR‐3 cells were transfected with si‐PACT # 1, si‐PACT # 2, and si‐PACT # 3. (A–D) A2780 and OVCAR‐3 cells were transfected with siRNA targeting PACT and the interference efficiency of si‐PACT was detected by RT‐qPCR and Western blot. (E–F) CCK‐8 was used to analyze the effect of PACT on the proliferation of A2780 and OVCAR‐3 cells. (G–H) EDU assay was used to analyze the effect of PACT on the proliferation of A2780 and OVCAR‐3 cells. (I–J) Transwell assay was used to analyze the effect of PACT on the migration of A2780 and OVCAR‐3 cells. (K–L) Transwell assay was used to analyze the effect of PACT on the invasion of A2780 and OVCAR‐3 cells. *p < 0.05 vs. control group; ^# p < 0.05 vs. si‐NC group.

3.3. Knockdown of PACT Triggered Ferroptosis in OC Cells

As shown in Figure 3A–D, transfection with si‐PACT significantly increased intracellular Fe²⁺ content and MDA levels in OVCAR‐3 cells compared with the control and si‐NC groups. Treatment with Fer‐1 (the ferroptosis inhibitor) alone reduced both parameters, and co‐treatment with si‐PACT and Fer‐1 partially reversed the si‐PACT‐induced elevation. PACT knockdown by si‐PACT significantly increased Lipid‐ROS levels in both A2780 and OVCAR‐3 cells compared with control and si‐NC groups, while Fer‐1 alone reduced Lipid‐ROS and co‐treatment with si‐PACT and Fer‐1 partially reversed this elevation (Figure 3E,G). Compared with the control group and si NC group, si PACT significantly reduced the expression of GPX4 and SLC7A11 in A2780 and OVCAR‐3 cells, and Fer‐1 treatment reduced their expression, reversing the effect of si‐PACT (Figure 3G,H). Additionally, si‐PACT decreased GPX4 activity and the GSH/GSSG ratio, both of which were restored by Fer‐1 (Figure 3I–L). The flow cytometry analysis revealed that PACT knockdown significantly increased the rate of cell death in both A2780 and OVCAR‐3 cells (Figure 3M,N). To determine whether si‐PACT‐induced cell death was primarily due to ferroptosis, cells transfected with si‐PACT#1 were treated with the ferroptosis inhibitor Ferrostatin‐1 (Fer‐1). The results showed that Fer‐1 effectively rescued the cell death triggered by PACT silencing (Figure 3M,N). Conversely, treatment with IKE (the ferroptosis inducer) significantly attenuated the anti‐ferroptotic effect of PACT overexpression in ovarian cancer cells (Figure S1).

Figure 3.

Interference with PACT triggered ferroptosis in OC cells. A2780 and OVCAR‐3 cells were transfected with si‐PACT and treated with iron death inhibitor Ferrostatin‐1 (Fer‐1, 1 μM). (A, B) The iron content of A2780 and OVCAR‐3 cells. (C, D) The MDA levels (E, F) Lipid‐ROS production (G, H) in A2780 and OVCAR‐3 cells. (G, H) The SLC7A11, GPX4 expression in A2780 and OVCAR‐3 cells. (I, J) The GPX4 content in A2780 and OVCAR‐3 cells. (K, L) The GSH/GSSG in A2780 and OVCAR‐3 cells. (M, N) The cell death of A2780 and OVCAR‐3 cells. *p < 0.05 vs. control group; ^# p < 0.05 vs. si‐NC group; ^& p < 0.05 vs. si‐PACT#1 group; ^$ p < 0.05 vs. Fer‐1 group.

3.4. Knockdown of PACT Regulated the Proliferation, Migration, and Invasion of A2780 Cells by Inhibiting TERT Expression

We next investigated the role of TERT in the PACT‐mediated malignant phenotype of A2780 cells. Results showed that PACT interference downregulated both mRNA and protein levels of TERT (Figure 4A,B). Overexpression of TERT not only elevated TERT expression but also reversed the suppression induced by si‐PACT (Figure 4A,B). Furthermore, TERT overexpression partially restored the decrease in telomerase activity caused by si‐PACT (Figure 4C). Functionally, TERT enhanced the proliferation, migration, and invasion of A2780 cells and attenuated the inhibitory effects of si‐PACT on these malignant phenotypes (Figure 4D–F). We further examined whether TERT participates in PACT‐regulated ferroptosis. TERT overexpression significantly reduced the intracellular iron content (Figure 4G), and decreased MDA and Lipid‐ROS levels (Figure 4H,I). It also increased the expression of SLC7A11 and GPX4 (Figure 4J). As shown in Figure 4K, compared with the control group, si‐PACT significantly reduced the GSH/GSSG ratio, overexpression of TERT alone increased the GSH/GSSG ratio, while co‐expression of ov‐TERT and si‐PACT greatly restored the ratio to near control levels. Figure 4L shows that the GPX4 content was significantly reduced after PACT knockout, while the co‐expression of ov‐TERT reversed the downregulation of GPX4 induced by si‐PACT. Similarly, Figure 4M shows that ov‐TERT reversed the promoting effect of si‐PACT on cell death. As expected, we obtained the same results in OVCAR3 cells (Figure S2).

Figure 4.

Interfering with PACT regulated the proliferation, migration, invasion, and ferroptosis of A2780 cells by inhibiting TERT expression. A2780 cells were transfected with si‐PACT or TERT overexpression plasmids. (A, B) RT‐qPCR and Western blot were used to detect the TERT mRNA and protein expression. (C) The telomerase activity of TERT in A2780 cells. (D) CCK‐8 was used to analyze the proliferation of A2780 cells. (E) Transwell assay was used to analyze the migration of A2780 cells. (F) Transwell assay was used to analyze the invasion of A2780 cells. (G) The iron content of A2780 cells. (H) MDA levels of A2780 cells. (I) Lipid‐ROS production of A2780 cells. (J) SLC7A11 and GPX4 expression in A2780 cells. (K) The GSH/GSSG in A2780 cells. (L) The GPX4 activity in A2780 cells. (M) The cell death rate of A2780 cells. *p < 0.05 vs. control group; ^# p < 0.05 vs. si‐PACT#1 group; ^& p < 0.05 vs. ov‐TERT group.

3.5. PACT Regulated TERT Expression Through Keap1‐Nrf2 Pathway

A2780 cells were transfected with ov‐PACT or si‐PACT#1. Overexpression of PACT led to reduced Keap1 expression and increased levels of Nrf2 and its downstream targets NQO1 and HO‐1, whereas PACT knockdown produced the opposite effects (Figure 5A). To examine whether PACT regulates Keap1 ubiquitination, co‐immunoprecipitation was performed, which revealed an interaction between PACT and Keap1 (Figure 5B). Ubiquitination assays indicated that Keap1 ubiquitination was markedly enhanced and its protein level decreased upon PACT overexpression, suggesting that PACT promotes Keap1 ubiquitination (Figure 5C). Cycloheximide (CHX) chase assays showed that PACT overexpression accelerated Keap1 protein degradation, an effect that was partially reversed by the proteasome inhibitor MG132 (Figure 5D,E). Furthermore, PACT overexpression reduced the binding of Keap1 to Nrf2 and facilitated Nrf2 nuclear translocation (Figure 5F,G). Figure 5H shows that ov‐PACT significantly increased TERT mRNA levels, and this induction was abolished by co‐transfection with si‐Nrf2. Finally, knockdown of Nrf2 abolished the upregulation of TERT and Nrf2 expression and telomerase activity induced by PACT overexpression, indicating that PACT enhances TERT expression through Nrf2 activation (Figures 5I,J). As expected, we obtained the same results in OVCAR3 cells (Figure S3).

Figure 5.

PACT promotes Nrf2 activation by mediating Keap1 ubiquitination and degradation. (A) Western blot analysis of Keap1, Nrf2, NQO1, and HO‐1 expression in A2780 cells transfected with ov‐PACT or si‐PACT#1. (B) Co‐immunoprecipitation assay demonstrating the interaction between PACT and Keap1. (C) Ubiquitination assay showing increased ubiquitination and decreased protein level of Keap1 in ov‐PACT group. (D, E) Keap1 protein degradation assay using CHX and rescue effect of MG132 in PACT‐overexpressing cells. (F) Co‐IP showing reduced Keap1‐Nrf2 binding upon PACT overexpression. (G) Nuclear translocation of Nrf2 detected by immunofluorescence and subcellular fractionation. (H) TERT mRNA expression in A2780 cells with Nrf2 knockdown and PACT overexpression. (I, J) TERT protein expression and telomerase activity in cells with Nrf2 knockdown and PACT overexpression. *p < 0.05 vs. control group; ^# p < 0.05 vs. ov‐PACT group; ^& p < 0.05 vs. si‐Nrf2 group.

3.6. Knockdown of PACT Inhibits OC Tumor Growth In Vivo

BALB/c nude mice were randomly divided into two groups and subcutaneously injected with A2780 cells without transfection/transfected with si‐PACT, experimental grouped as si‐NC and si‐PACT. After 1 month, it was found that the tumor volume and weight in the si‐PACT group were smaller than those in the si‐NC group (Figure 6A–D). Interference with PACT significantly reduced the expression level of PACT protein in mouse tumor tissue, while increasing the level of Keap1 protein and inhibiting the expression of Nrf2 and TERT (Figure 6E). Compared with the si‐NC group, the telomerase activity in the si‐PACT group was significantly reduced (Figure 6F). MDA, and ROS levels in the tumor tissue of the si‐PACT group were significantly increased compared with the si‐NC group, the iron content, while the protein expression levels of GPX4 and SLC7A11 were significantly decreased (Figure 6G–J). Inhibition of PACT expression promoted ferroptosis in OC cells in vivo.

Figure 6.

Interference with PACT inhibits OC tumor growth in vivo. BALB/c nude mice were randomly divided into two groups and subcutaneously injected with A2780 cells without transfection/transfected with si‐PACT. Experimental grouping: si‐NC、si‐PACT. (A–B) The tumor in the si‐NC and si‐PACT group. (C–D) The tumor volume and weight in the si‐NC and si‐PACT group. (E) The protein expression levels of PACT, Keap1, Nrf2, and TERT in tumor tissues. (F) The telomerase activity in tumor tissues. (G) The level of ROS in tumor tissues. (H) The iron content in tumor tissues. (I) The level of MDA in tumor tissues. (J) The protein expression levels of GPX4 and SLC7A11 in tumor tissues. *p < 0.05 vs. si‐NC group.

4. Discussion

Recent studies found that PACT is abnormally expressed and plays a critical role in various cancers [19]. For example, Li et al. found that PACT is highly upregulated in oesophagal cancer, and knockdown of PACT reduced p53 polyubiquitination, enhanced p53 accumulation, led to cell apoptosis, and inhibited cell growth [20]. In a study on cervical cancer, the increase of PACT promoted cell viability and migration, thereby exacerbating the malignant progression of the cancer [21]. PACT is also involved in regulating the progression of OC. In addition, PACT, as a target molecule of miR‐107 and miR‐193a‐5p, participates in regulating the malignant biological behavior of OC cells [22, 23]. Similarly, previous studies have reported that PACT is highly expressed in OC and stimulates proliferation and migration potential by targeting PIK3R6 [24]. Importantly, in our preliminary study, we found that PACT was upregulated in OC tumor tissues and cell lines, as well as interfering with PACT inhibited the proliferation, migration, and invasion of OC cells and promoted ferroptosis. However, we found that the extent of the effect of interfering with PACT on the functions of A2780 and OVCAR‐3 cells was inconsistent, suggesting that it might be related to the origin, physiological characteristics, or malignancy of the cells.

The length of chromosome telomeres is closely related to the occurrence and development of tumors [25]. TERT, as the main active subunit of telomerase, has long been known as an “anti‐aging molecule” due to its classic telomere length maintenance function. TERT expression and telomerase activity are reactivated in 90% of malignant tumors [26]. Research reports that TERT is highly expressed in various malignant tumors, such as thyroid cancer, glioma, urothelial carcinoma, melanoma, and liver cancer, and is associated with poor prognosis [27]. Yu et al. found that TERT mutations make thyroid cancer prognosis worse and more prone to recurrence [28]. In addition, approximately 75% of melanomas contain mutations in the TERT gene promoter, which can activate telomerase production and increase telomerase activity, promoting cancer progression [29]. In addition, TERT and telomerase are closely related to ferroptosis. In animal models of acute lung injury, low TERT levels enhance ferroptosis, while overexpression of TERT alleviates ferroptosis [30]. In addition, telomerase inhibitors can also accelerate cell iron death [31]. Interestingly, telomerase inhibitors induce ferroptosis and have clinical efficacy in treating myelofibrosis and myelodysplastic syndrome, indicating that TERT and telomerase may contribute to cancer progression through the ferroptosis pathway [32]. Therefore, the study of the oncogenic mechanism of TERT reactivation and telomerase is a new direction in oncology research, and the development of effective treatment strategies targeting TERT has significant clinical significance.

In this study, we found that knocking down PACT reduced the mRNA and protein expression levels of TERT, and interfering with PACT led to a decrease in telomerase activity. It can be seen that PACT regulates the expression of TERT. We all know that PACT is a non‐binding protein, which means it does not regulate TERT expression through direct interaction with the TERT promoter. A large amount of evidence indicates that E3 ligases can bind to transcription factors on the TERT promoter and alter their expression. Mosweu et al. reported that PACT indirectly affected the expression of TERT by interacting with certain activation and/or inhibition factors on the TERT promoter through its E3 ligase activity [18]. Based on this, we speculate that PACT may also affect the expression of TERT in this way in our research, but drawing the conclusion must be cautious, and further exploration is needed.

Our findings demonstrate that PACT promotes OC progression by enhancing Keap1 ubiquitination and degradation, leading to Nrf2 activation and nuclear translocation, which aligns with established roles of the Keap1‐Nrf2 pathway in cancer. Persistent Nrf2 activation, often resulting from Keap1 dysfunction or mutation, drives tumor proliferation, metastasis, and therapy resistance by upregulating antioxidant and detoxification genes (HO‐1, NQO1) [33]. For instance, in endometrial cancer, Keap1 downregulation enhances Nrf2 nuclear accumulation, increasing chemoresistance, while Keap1 inhibitors like CB5712809 exacerbate Nrf2‐driven tumor growth in colon cancer [34]. Nrf2 nuclear translocation further supports metabolic reprogramming and oxidative stress adaptation, as observed in lung and OC s, where Nrf2 activation reduces ROS and promotes survival [35]. Thus, our results reinforce that PACT‐mediated Keap1‐Nrf2 disruption contributes to OC pathogenesis, echoing broader cancer biology mechanisms. We acknowledge that direct evidence linking PACT to Nrf2 ubiquitination status is not provided in the current study, and we plan to investigate this aspect in future experiments.

In conclusion, the study demonstrates that PACT plays a critical oncogenic role in OC by promoting proliferation, migration, and invasion while suppressing ferroptosis. Mechanistically, PACT facilitates Keap1 ubiquitination and degradation, leading to Nrf2 activation and subsequent nuclear translocation. This enhances TERT expression and telomerase activity, driving malignant behaviors and resistance to ferroptosis. Genetic or pharmacological inhibition of PACT restores ferroptosis sensitivity and suppresses tumor growth in vivo. These findings identify the PACT/Keap1/Nrf2/TERT axis as a key regulatory pathway and suggest PACT targeting as a potential therapeutic strategy for OC.

Author Contributions

Xiaofeng Yang: conceptualization, methodology, and writing – reviewing and editing. Qian Zhang and Ting Yang: data curation and formal analysis. Jing Zhang and Xiaofei Tian: formal analysis and software. Zhihong Han: data curation and software. Xuemei Yang: writing – original draft, methodology, and resources.

Ethics Statement

This study was formally approved by the ethics committee of the First Affiliated Hospital of Xi'an Jiaotong University. All animal experiments complied with ARRIVE guidelines and were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: PACT overexpression attenuates ferroptosis in OC cells. A2780 and OVCAR‐3 cells were transfected with pcDNA3.1‐PACT (ov‐PACT) and treated with IKE (the ferroptosis inducer). (A, B) The iron content of A2780 and OVCAR‐3 cells. (C, D) The MDA levels (E, F) Lipid‐ROS production (G, H) in A2780 and OVCAR‐3 cells. (G, H) The SLC7A11, GPX4 expression in A2780 and OVCAR‐3 cells. (I, J) The GPX4 activity in A2780 and OVCAR‐3 cells. (K, L) The GSH/GSSG in A2780 and OVCAR‐3 cells. (M, N) The cell death of A2780 and OVCAR‐3 cells. *p < 0.05 vs. control group; ^# p < 0.05 vs. ov‐NC group; ^& p < 0.05 vs. ov‐PACT group; ^$ p < 0.05 vs. IKE group.

Figure S2: Interfering with PACT regulated the proliferation, migration, invasion, and ferroptosis of OVCAR3 cells by inhibiting TERT expression. OVCAR3 cells were transfected with si‐PACT or TERT overexpression plasmids. (A, B) RT‐qPCR and Western blot were used to detect the TERT mRNA and protein expression. (C) The telomerase activity of TERT in OVCAR3 cells. (D) CCK‐8 was used to analyze the proliferation of OVCAR3 cells. (E) Transwell assay was used to analyze the migration of OVCAR3 cells. (F) Transwell assay was used to analyze the invasion of OVCAR3 cells. (G) The iron content of OVCAR3 cells. (H) MDA levels of OVCAR3 cells. (I) Lipid‐ROS production of OVCAR3cells. (J) SLC7A11 and GPX4 expression in OVCAR3 cells. (K) The GSH/GSSG in OVCAR3 cells. (L) The GPX4 activity in OVCAR3 cells. (M) The cell death rate of OVCAR3 cells. *p < 0.05 vs. control group; ^# p < 0.05 vs. si‐PACT#1 group; ^& p < 0.05 vs. ov‐TERT group.

Figure S3: PACT promotes Nrf2 activation by mediating Keap1 ubiquitination and degradation in OVCAR3 cells. (A) Western blot analysis of Keap1, Nrf2, NQO1, and HO‐1 expression in OVCAR3 cells transfected with ov‐PACT or si‐PACT#1. (B) Co‐immunoprecipitation assay demonstrating the interaction between PACT and Keap1. (C) Ubiquitination assay showing increased ubiquitination and decreased protein level of Keap1 in ov‐PACT group. (D, E) Keap1 protein degradation assay using CHX and rescue effect of MG132 in PACT‐overexpressing cells. (F) Co‐IP showing reduced Keap1‐Nrf2 binding upon PACT overexpression. (G) Nuclear translocation of Nrf2 detected by immunofluorescence and subcellular fractionation. (H) TERT mRNA expression in OVCAR3 cells with Nrf2 knockdown and PACT overexpression. (I, J) TERT protein expression and telomerase activity in cells with Nrf2 knockdown and PACT overexpression. *p < 0.05 vs. control group; ^# p < 0.05 vs. ov‐PACT group; ^& p < 0.05 vs.si‐Nrf2 group.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.