OCT4 induces EMT and promotes ovarian cancer progression by regulating the PI3K/AKT/mTOR pathway

Abstract

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 benign cyst tissues, OC tissues, and omental metastatic tissues were performed to reveal 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 knocked down to assess the proliferation, migration, and invasion of OC cells using CCK-8, colony formation, wound healing, and Transwell assays. In addition, the nude tumor mouse model was used for 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. The interaction between OCT4 and p-AKT was further confirmed by coimmunoprecipitation (CoIP) assays. Importantly, AKT activation by its activator SC79 reversed the biological functions of OCT4 knockdown.

Results: OCT4 expression was significantly upregulated in OC samples and metastatic tissues. OCT4 knockdown 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. An AKT agonist reversed the effect of OCT4 knockdown on OC cells. 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.

Keywords: AKT; OCT4; invasion; migration; ovarian cancer; proliferation.

Figure 1

OCT4 IHC staining and survival analysis (A) Representative IHC staining of OCT4 in benign ovarian cyst tissues, OC tissues, and matched omental metastatic tissues. (B) Quantification of OCT4 H-score in different tissues. (C) OS of OC patients with low/high OCT4 H-score. (D) Expression rate of OCT4 in OC with or without lymph node metastasis. (E) Expression rate of OCT4 in high/low histological grade OC. (Data were expressed as the mean ± SD. Significance was calculated using Student’s t-test. ****, P<0.0001.)

Figure 2

Expression of OCT4 in OC cell lines (A, B) OCT4 protein expression in OC cell lines compared with normal ovarian epithelial cell lines (IOSE80). (C, D) OCT4 protein expression in SKOV3 and A2780 cells transfected with shRNAs targeting OCT4 by Western blotting assay. (E, F) The above expression was verified by IF analysis. (Data were expressed as the mean ± SD. Significance was calculated using Student's t-test. ****, P<0.0001.)

Figure 3

Knockdown of OCT4 inhibited the proliferation of OC cells (A, B) In SKOV3 and A2780 cell lines, cell viability was measured by CCK-8 assay. (C, D) Colony formation assays and statistical analysis in SKOV3 and A2780 cell lines. (Data were expressed as the mean ± SD. Significance was calculated using Student's t-test. *P<0.05, ****P<0.0001).

Figure 4

Knockdown of OCT4 inhibited the migration and invasion of OC cells (A, B) In two cell lines, wound healing assays indicated the percent of wounds closed. (C, D) Transwell assays were used to measure the migration and invasion of two cell lines. (E–G) Quantification of cell migration and invasion was measured by wound healing or Transwell assays. (Data were expressed as the mean ± SD. Significance was calculated using Student’s t-test. **P<0.01, ****P<0.0001).

Figure 5

OCT4 influenced the PI3K/AKT/mTOR signaling pathway in OC cells. (A, B) Western blotting was used to measure representative proteins and their relative expression levels in the PI3K/AKT/mTOR pathway. In addition, OCT4 knockdown inhibited the expression of the EMT pathway. (C, D) IF assays detected the relationship between OCT4 and p-AKT in OC cell lines. (E) CoIP of OCT4 with p-AKT in A2780 cell lines. The input represents the total protein extract used in IP. IB, immunoblotting; IP, immunoprecipitation. (Data were expressed as the mean ± SD. Significance was calculated using Student's t-test. ***P<0.001, ****P<0.0001).

Figure 6

OCT4 modulated proliferation, migration and invasion by activating the AKT signaling pathway in OC cells. (A, B) In SKOV3 and A2780 cell lines, CCK-8 assay data showed that SC79 treatment rescued the inhibition of proliferation by OCT4 knockdown. (C–F) Transwell assays indicated that the suppression of migration and invasion ability by OCT4 knockdown was rescued after SC79 treatment. (Data were expressed as the mean ± SD. Significance was calculated using Student’s t-test. *P<0.05, ****P<0.0001.)

Figure 7

The effect of OCT4 knockdown on the proliferation and metastasis of OC in vivo. (A) The subcutaneous injection model of nude mice. (B, C) Size of tumor-bearing nude mice in different groups. (D) Tumor volume was measured weekly after injecting tumor cells. (E) Quantification of tumor weight. (F) Analysis of the location and number of metastases in tumor-bearing mice of different groups. (G) HE staining of ovarian neoplasms and metastatic tissues. (H) IHC assays of OCT4 and p-AKT in mouse tumors. (I) Model diagram showing the role of OCT4 and the PI3K/AKT/mTOR pathways as well as the EMT pathways regulating the proliferation and metastasis of OC cells. (Data were expressed as the mean ± SD. Significance was calculated using Student’s t-test. *P<0.05, ***P<0.001.).