OCT4 Promotes Ovarian Cancer Progression by Regulating the PI3K/AKT/mTOR Pathway


 Background: Octamer-binding transcription factor 4 (OCT4) is a key stem cell transcription factor involved in the development of various cancers. The role of OCT4 in ovarian cancer (OC) progression and its molecular mechanism are not fully understood.Methods: First, immunohistochemistry (IHC) assays of ovarian normal tissues, OC samples, and metastatic tissues were performed to reveal the OCT4 expression profiles. We knocked down OCT4 in two OC cell lines (SKOV3 and A2780) using a lentiviral vector and performed in vitro and in vivo experiments. OCT4 was silenced to assess the proliferation, migration, and invasion of OC cells using CCK-8, colony formation, wound healing, and Transwell asssays. In addition, a nude tumor mouse model was used for the in vivo study. Mechanistically, we demonstrated that OCT4 influenced protein expression in the phosphoinositol 3-kinase (PI3K)/AKT/mTOR pathway and epithelial-mesenchymal transition (EMT)-related proteins by Western blotting and immunofluorescence (IF) assays.Results: OCT4 expression was significantly upregulated in OC samples and metastatic tissues. OCT4 silencing notably inhibited the proliferation, migration, and invasion of OC cells in vitro and in vivo. Moreover, the expression of p-PI3K, p-AKT, and p-mTOR was downregulated after OCT4 knockdown. EMT in OC samples was enhanced by OCT4.Conclusions: Our study shows that OCT4 promotes the proliferation, migration, and invasion of OC cells by participating in the PI3K/AKT/mTOR signaling axis, suggesting that it could serve as a potential therapeutic target for OC patients.


Background
Over the past four decades, cancer survival has improved for the most common cancers (1). Despite these advances, OC is the fth most frequent cause of cancer-related death in women, with the highest casefatality rate among gynecologic malignancies (2). The American Cancer Society estimates that 13,940 OC-related deaths (5% of all cancer cases) occurred in the United States in 2021 (3). Although this disease is highly curable in the early stage, there is a lack of effective screening options at the early stage, most patients present with stage III/IV disease, and more than 75% of women with advanced OC die from the disease (4). Cytoreductive surgery and combined platinum-taxane chemotherapy have been considered the mainstay of therapy for decades (5). Therefore, emphasizing the importance of potential biomarkers for early diagnosis and timely specialist treatment is increasingly urgent.
OCT4, an octamer binding transcription factor 4, also known as POU5F1 and OCT3, is a member of the POU family of transcription factors (TFs). Its main function is to bind the octamer sequence motif (ATGCAAAT) to activate the expression of target genes (6). Gene expression, protein modi cation, stability, and activity of OCT4 are strictly regulated during embryonic development (7). OCT4 is undetectable in the cells of mature organisms, so its re-expression is closely related to tumor development and progression (8). Studies have shown that high OCT4 expression is signi cantly associated with decreased overall survival (OS) in patients with pancreatic cancer (9). In gastric cancer cells, OCT4 and SOX2 promote tumor proliferation, migration, invasion, and tumorigenicity, and the two genes may have synergistic effects to some extent (10). Nevertheless, the role of OCT4 in tumorigenesis has not been con rmed.
In this study, IHC assays were performed to analyze the expression pro le of OCT4 by using a tissue microarray (TMA) of our samples. Two cell lines (SKOV3 and A2780) were used to detect the function of OCT4 and its mechanism in OC progression. Our ndings may provide new directions for the diagnosis and treatment of OC.

Patients and specimens
Ovarian TMAs were obtained from patients diagnosed between 2008 and 2018 at Shanghai Jiaotong University School of Medicine Xinhua Hospital after obtaining the participants' informed consent. OC samples veri ed by postoperative pathology were included in the study, while patients with other malignant tumors were excluded. The histopathological diagnosis, grade, and stage of OC were based on the FIGO classi cation. This study was approved by the Ethics Committee of Xinhua Hospital.

Cell lines and culture
The OC cell lines SKOV3 and A2780 were cultured in Dulbecco's modi ed Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco, USA), penicillin (100 U/ml) and streptomycin (100 ng/ml). All cell lines were purchased from the American-type culture collection (ATCC, Manassas, VA, USA) and cultured at 37℃ in a 5 % CO 2 atmosphere. All cell lines were authenticated by STR pro ling and tested regularly for mycoplasma contamination.

Western blotting assay
Cells were lysed in RIPA buffer, and lysates were separated by 6%-10% SDS-PAGE. Then, the proteins were transferred onto polyvinylidene uoride membranes (Millipore, IPVH00010). After the membranes were blocked with 5% BSA for 2 h, they were incubated with primary antibodies overnight at 4 °C and washed with 1×Tris-buffered saline with Tween 20 (TBST). Subsequently, the membranes were incubated with a secondary HRP-conjugated antibody for 2 h at room temperature (RT) and washed with TBST. Finally, the signals were visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore, US). The antibodies used are listed in Supplementary Table S1.

IF assay
Cells were xed in 4% paraformaldehyde for 30 min, permeated with 2% Triton X-100 for 10 min, and sealed in 5% goat serum for 60 min at RT. Subsequently, the samples were incubated at RT with primary and secondary antibodies for 60 min. The secondary antibodies conjugated with Alexa Fluor 594 (Molecular Probes, USA) were then restained at RT for 30 min. Nuclei were restained with 4′,6-diamidino-2phenylindole dihydrochloride (DAPI, Life Technologies, USA) at RT for 5 min. IF signals were captured using a Leica SP5 confocal uorescence microscope (Wetzlar, Frankfurt, Germany).

IHC
TMAs were conducted using benign ovarian tumors, OC primary lesions, OC metastatic lesions, and an OCT4 antibody (ab181557, Abcam, 1:100 dilution). According to the dyeing depth, the intensity of staining was classi ed as weak, moderate, and strong. Protein expression was semiquanti ed using the histochemistry score (H-score), which was calculated as ∑ (PI × I) = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + (percentage of cells of strong intensity × 3). The nal score was calculated as the average H-score of the duplicate tissue microarray for each tissue type.

Cell Counting Kit (CCK)-8 proliferation assay
A CCK-8 (Beyotime, Shanghai, China, Cat# C0039) assay was used to determine OC cell viability. Cells were seeded in 96-well plates at a density of 2000 cells per well. After culture for 1, 2, 3, 4, and 5 days, 100 ul of medium containing 10 µl of CCK-8 reagent was added, and the cells were incubated at 5% CO 2 and 37 ˚C for 2 h. The absorbance was measured at a wavelength of 450 nm using a plate reader.
Colony formation assay SKOV3 and A2780 cells were cultured in 6-well plates at 1000 cells per well. They were cultured at 5% CO 2 and 37℃ for at least 7 days. The colonies were stained with 4% paraformaldehyde for 20 min and 0.2% crystal violet for 15 min and counted.
Wound healing assay Cells were seeded in 6-well plates at 9×10 5 cells per well as con uent monolayers. A single layer was wounded in the middle of the well with a standard pipette tip. Subsequently, the scratches were washed with phosphate-buffered saline (PBS) to remove cellular debris. After incubation for 24 h, the area of the cell-free wound was examined with microscopy.

Migration and invasion assays
Cell migration and invasion were evaluated using a Transwell chamber (8 μm pore; Corning, 3422). Cell suspensions (6 × 10 4 cells) in serum-free DMEM were added to the upper chamber (for migration assays) or the chamber precoated with Matrigel (for invasion assays). DMEM containing 10% FBS was added to the lower chamber. After incubation for 24 h, the cells that invaded or migrated the lower chamber were stained with 0.2% crystal violet for 15 min. Five random images of different elds of vision under a microscope (Olympus) were captured, and the number of migrated or invaded cells was counted.

Animal tumor model
Animal care and experimental procedures were performed following Guidelines for Animal Experiments and were approved by the Animal Care and Use Committee of Xinhua Hospital in our study. Five-week-old female BALB/c nude mice (Department of Laboratory Animals, Xinhua Hospital) were randomly divided into different groups (n=5) for subcutaneous injection with A2780-control, A2780-OCT4-sh1, and A2780-OCT4-sh2 cells (2×10 6 cells for each mouse). The tumor volume was calculated using the following formula: volume =width 2 ×length×0.52. After subcutaneous injection, tumor development was assessed weekly. Five weeks later, the tumors in each group were harvested after euthanasia, and the weights of the tumors and the number of metastatic organs were recorded. Tumor tissues and metastatic organs were xed with 4% neutral buffered formalin for frozen section preparation.

Statistical analysis
The data in this study were calculated using SPSS 24.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism (Version 8.0, San Diego, CA, USA) and are presented as the mean ± standard deviation (SD). Student's t test, two-way ANOVA, and chi-square tests were used for comparisons between groups. Each experiment was repeated at least twice. Subsequent multivariate analysis based on the Cox proportional hazard model included variables with univariate analysis values of P <0.05. A probability less than 0.05 was considered signi cant. Kaplan-Meier survival analysis was used to evaluate the correlation between clinical outcome and gene expression, and the P value was calculated by the log-rank test.

OCT4 expression was signi cantly upregulated and was closely related to clinicopathological features in OC
To determine whether OCT4 expression is correlated with OC progression, we collected ovarian benign tumor tissues (n=79), OC primary lesion tissues (n=116), and OC metastatic lesion tissues (n=71) in the greater omentum and examined OCT4 expression by using IHC. The OCT4 H-score of the primary and metastatic tissues was signi cantly higher than that of the ovarian benign tissues ( Figure 1A-B). In addition, OCT4 expression was further increased in the metastatic tissues compared with the primary OC tissues ( Figure 1A-B). To investigate the clinical signi cance of OCT4 in OC, we collected the medical history of 116 OC patients and analyzed the association between OCT4 expression and the clinicopathologic characteristics of OC patients ( Table 1). The boundary between high-level and low-level expression of OCT4 was the median H-score. As shown in Table 1, OCT4 expression was signi cantly correlated with histological grade and lymph node metastasis ( Figure 1D-E), while there was no signi cant association between OCT4 expression and age, FIGO stage, or histological subtype. Following information about patient outcomes, survival analysis showed that high expression of OCT4 was signi cantly associated with poorer OS ( Figure 1C). Taken together, these data suggested that upregulated expression of OCT4, as an independent prognostic factor, had a signi cant correlation with poor prognosis of OC and may contribute to OC progression.

OCT4 protein expression was upregulated in the OC cell lines SKOV3 and A2780
Compared with that in normal ovarian epithelial cell lines (IOSE80), OCT4 was highly expressed in OC cell lines (SKOV3, A2780, OVCA433, and SKOV3-ip1), whereas its expression was relatively low in HEY, HEY A8, ES-2, and OVCA429 cells (Figure 2A-B). OCT4 expression was particularly high in two invasive cell lines, SKOV3 and A2780, compared with other cell lines. To further explore the regulatory role of OCT4 in OC, we constructed stable OCT4 knockdown cell lines (OCT4-sh1 and OCT4-sh2) and the control vector cell lines (Control) by lentiviral transfection in SKOV3 and A2780 cells, which was veri ed by Western blotting assays (Figure 2C-D) and IF analysis ( Figure 2E-F).
Knockdown of OCT4 inhibited the proliferation of OC cells Next, we tested the effect of OCT4 knockdown on the proliferation by CCK-8 assays and found that OCT4 knockdown signi cantly reduced cell growth ( Figure 3A-B). Colony formation assays indicated that OCT4 silencing decreased the size and number of colonies of SKOV3 and A2780 cells ( Figure 3C-D).

Knockdown of OCT4 inhibited the migration and invasion of OC cells
By performing the wound healing assay, we found that the attening and spreading range was decreased in the SKOV3/OCT4-sh1/2 and A2780/OCT4-sh1/2 cells, respectively, compared with the controls ( Figure  4A-B, E). Transwell assays revealed that cell migration and invasion were signi cantly inhibited in the OCT4 knockdown cells compared with the control cells ( Figure 4C-D, F-G).

Knockdown of OCT4 inactivated the PI3K/AKT/mTOR pathways in OC cells
The PI3K/AKT/mTOR signaling pathway is one of the most frequently identi ed pathways in human cancer and plays a key role in driving tumor initiation and progression (11). To explore the downstream signaling pathway regulated by OCT4, we used Western blotting to detect the association between OCT4 and the PI3K/AKT/mTOR signaling pathway. OCT4 silencing inhibited the accumulation of p-PI3K, p-AKT, and p-mTOR proteins, suggesting that OCT4 may regulate the progression of OC through the PI3K/AKT/mTOR signaling pathway ( Figure 5A-B). In addition, we performed an IF assay, which further supported the colocalization between OCT4 and p-AKT ( Figure 5C-D). Loss of E-cadherin gene expression promotes dysfunction of the cell connection system, leading to invasion and metastasis of cancer cells, which has been considered the most important feature of EMT. Western blotting of the EMT markers Ecadherin and N-cadherin in the SKOV3-OCT4-sh1/sh2 and A2780-OCT4-sh1/sh2 cell lines showed that OCT4 silencing inhibited EMT ( Figure 5A-B).

Knockdown of OCT4 inhibited the proliferation and metastasis of OC in vivo
To further con rm the role of OCT4 in vivo, we established a xenograft model in nude mice using the A2780 cell line ( Figure 6A). The model results showed that tumor growth was signi cantly inhibited by OCT silencing (Figure 6B-C). OCT4 silencing reduced the volume and weight of the A2780 cell-derived tumors ( Figure 6D-E). The mice inoculated with A2780-OCT4-Control developed more metastases, while fewer metastases were observed in the A2780-OCT4-sh1/sh2 mouse tumor models ( Figure 6F). HE staining was performed on the right ovarian, liver, and spleen metastases of the tumor-bearing mice ( Figure 6G). We conducted IHC assay on the mouse tumors and found that the OCT4-sh1/sh2 group had reduced the expression levels of p-AKT ( Figure 6H). Finally, we identi ed the roles of OCT4 and the PI3K/AKT/mTOR pathways as well as the EMT pathways in the regulation of proliferation and metastasis of OC ( Figure 6J).

Discussion
OCT4 promoted the formation and progression of cancer and was associated with poor clinical outcomes (12)(13)(14)(15). OCT4 expression has been detected in various types of malignant neoplasms, and increased expression levels are associated with advanced tumor grade, metastatic formation, and survival rate, underscoring the clinical relevance of OCT4 (14). Several studies have shown that OCT4 expression is upregulated in head and neck squamous carcinoma (12), bladder cancer (13,16), lung cancer (14), breast cancer (17), oral squamous cell carcinoma (18), and cervical cancer (19).
In this study, we performed IHC staining and survival analysis on primary and metastatic OC tissues to prove that OCT4 can be regarded as a prognostic risk factor to induce OC progression and metastasis.
OCT4 was signi cantly elevated in OC tissues and cell lines. We constructed two OC cell lines with OCT4 knockdown using stable lentivirus strains. Through CCK8, colony formation, wound healing, and Transwell assays, we found that OCT4 signi cantly promoted cell proliferation and invasion in vitro. Next, we established a xenograft model in nude mice using A2780 cells to demonstrate that OCT4 promotes tumor progression and metastasis in vivo.
The core focus of tumor research has been to detect tumor markers and identify directions for targeted therapies, especially after the tremendous efforts of The Cancer Genome Atlas Project (TCGA) sequencing. Our current understanding of OCT4 function supports its importance in tumorigenesis, where its presence is associated with poorer prognosis in most cancer types. However, the mechanism and pathway of action remain unknown.
PI3K is one of the most frequently altered pathways in human malignancies, controlling most characteristics of cancer, including cell proliferation, migration, metastasis, and survival (20). AKT is a serine-threonine kinase, a known PI3K target that regulates numerous downstream target genes (21). To further investigate whether the PI3K/AKT/mTOR signaling pathway was involved in the oncogenic mechanism of OCT4 in OC, we evaluated the relationship between OCT4 and p-PI3K, p-AKT, or p-mTOR. OCT4 promoted the activation of the PI3K/AKT/mTOR signaling pathway in OC. EMT is considered a pathological process that leads to tumor progression and is related to invasion and metastasis. In nasopharyngeal carcinoma, OCT4 is located in the anterior area of tumor invasion and is signi cantly associated with various invasive behaviors and EMT (22). As expected, we detected increased E-cadherin and reduced N-cadherin expression in OCT4 knockdown cell lines.

Conclusion
OCT4 expression is upregulated in OC, but this is the rst study to show that OCT4 promotes proliferation and metastasis by affecting the PI3K/AKT/mTOR pathway in OC cells. Our research showed that OCT4, as an independent predictor, was signi cantly associated with poor prognosis and contributed to the progression of OC.