OCT4 Promotes Ovarian Cancer Cell Metastasis and Angiogenesis via Modulating VEGFR2/LRPPRC Pathway

Background: Due to its high ability of metastasis, ovarian cancer remains the most lethal gynecological malignancy, yet its underlying mechanism remains unconrmed. Objectives: The main purpose is to probe into the role and regulation mechanism of octamer-binding transcription factor 4 (OCT4) in angiogenesis and metastasis in ovarian cancer. Methods: Immunohistochemistry (IHC) and immunouorescence in epithelial ovarian cancer specimens and benign ovarian tumor samples were performed, followed by RNA-sequencing and examination of angiogenesis, cell migration and invasion in OCT4 knockdown cell lines and the controls. Co-Immunoprecipitation (Co-IP), mass spectrometry, immunoblotting, immunouorescence and chromatin immunoprecipitation (ChIP) analyses were conducted along with models of zebrashes and nude mice of transplanted tumors to gain insights into the specic functions and mechanisms of action of OCT4 in ovarian cancer. Results: Firstly, we discovered that OCT4 expression was enhanced in ovarian cancer tissues signicantly, especially in the metastatic lesions, indicating that OCT4 might be a key for the metastasis of ovarian cancer. Furtherly, we observed and veried that OCT4 promoted cell migration and invasion, and induced angiogenesis in vitro and in vivo. Mechanistically, OCT4 modulated the transcription of leucine ‐ rich PPR motif ‐ containing protein (LRPPRC)(cid:0)and furtherly interacted with vascular endothelial growth factor receptor 2 (VEGFR2)/LRPPRC complex and ultimately triggered the downstream FAK/AKT signaling pathway . Conclusions: Together, through models of ovarian cancer cells, zebrashes and tumor-transplanted mice, this study highlighted the importance of OCT4/LRPPRC/VEGFR2 signaling axis in metastasis of ovarian cancer and angiogenesis. Thus, our nding supplied a potential novel molecular-targeted approach for the treatment of ovarian cancer.


Introduction
With metastatic state of 70% patients at diagnosis, ovarian cancer remains the gynecological malignancy with highest mortality, though there were progression in the therapeutic modes, including surgery and chemotherapy [1,2]. As is reported, the 5-year survival rate of ovarian cancer patients is only 30.2% [3].
However, the mechanism of action of ovarian cancer progression and metastasis remains uncertain. The objective of our study is to discover key factors participating in cell metastasis and probe into the molecular mechanism.
Octamer-binding transcription factor 4 (OCT4), also named as pit-oct-unc (POU) domain class 5 transcription factor 1 (POU5F1), is a noted regulatory factor of self-renewal and cell differentiation in stem cells [4,5]. It was rst reported in 1989 [6], and its expression was discovered in ovulated oocytes, inner cell mass, embryonic stem cells, embryonic germ cells, and embryonic carcinoma cells [7].
Nowadays, OCT4 has been detected in several sorts of human cancers and observed to play an essential role in carcinogenesis. For example, OCT4 was enhanced in breast cancer [8], colorectal cancer [9,10], liver cancer [11], and ovarian cancer [12]. Moreover, OCT4 could promote prostate cancer progression [13] and liver metastasis of primary colorectal cancer [9], and accelerate tumorigenesis through activating JAK/STAT signaling in ovarian cancer [14]. However, function of OCT4 in the metastasis of ovarian cancer and its underlying molecular mechanisms remain uncertain.
Firstly, our study revealed that OCT4 was up-regulated in epithelial ovarian cancer patients compared with the benign ovarian tumors, especially in the metastatic lesions. Functionally, OCT4 promoted angiogenesis and ovarian cancer cell metastasis in vitro and in vivo. Mechanistically, OCT4 induced the transcription of leucinerich PPR motifcontaining protein (LRPPRC), and furtherly interacted with vascular endothelial growth factor receptor 2 (VEGFR2)/LRPPRC complex, and ultimately triggered its downstream FAK/AKT pathway. Accordingly, we proposed that OCT4 played a hitherto unrecognized mechanism in metastasis of ovarian cancer, and the novel OCT4/LRPPRC/VEGFR2 signaling axis might be effectively used for a promising therapeutic target.

Results
OCT4 was markedly enhanced in metastatic ovarian cancer tissues and might serve as an oncogene to lead to poor survival in the patients Firstly, we constructed 8 three-dimensional (3D) cultured organoids of epithelial ovarian cancer and performed immuno uorescence, and ultimately discovered that OCT4 was high expressed in ovarian cancer organoids (Fig. 1A). Then, via immuno uorescence staining assay in 20 primary ovarian cancer tissues and 20 metastatic specimens from greater omenta, and we discovered that the metastatic specimens exhibited higher expression of OCT4 compared with the primary lesions (Fig. 1B). Furtherly, we established tissue chips using 153 benign ovarian tumor tissues, 187 primary lesions of ovarian cancer, and 154 metastatic specimens from greater omenta. Through immunohistochemistry (IHC) technique and image analysis of these chips we discovered that OCT4 was high expressed in ovarian cancer tissues, especially in the metastatic sites (Fig. 1C). H-score of IHC evaluation was shown in Fig. 1D, and the statistics demonstrated that OCT4 high expression ration in metastatic lesions of ovarian cancer was obviously higher than that in primary specimens (81.82% vs. 63.64%), and both of them were higher than benign ovarian tumor tissues (18.95%, Fig. 1E). Furthermore, results of survival analysis showed that OCT4 high expression was signi cantly associated with shorter survival (OS and PFS) in ovarian cancer patients ( Fig. 1F-G). Together, above results demonstrated that OCT4 might serve as an oncogene to participate in ovarian cancer metastasis, and an enhanced OCT4 induced ovarian cancer patients' poor prognosis.

OCT4 elevated cell migration and invasion in ovarian cancer
To further probe into the modulation role of OCT4 in ovarian cancer, we established two kinds of OCT4targeting shRNAs and the control vector into ovarian cancer cell lines (OCT4-silencing ovarian cancer cell lines: SKOV3ip1/OCT4-sh1/2 and OVCA433/OCT4-sh1/2; control ovarian cancer cell lines: SKOV3ip1/Vector and OVCA433/Vector), which was proved by qRT-PCR and immunoblotting assay ( Fig.  2A-B). Next, we performed RNA-sequencing in SKOV3ip1/OCT4-sh1 ovarian cancer cells and the controls, and found that a number of differentially expressed genes (DEGs) were signi cantly altered (Fig. 2C).
Gene ontology (GO) analysis of biological process indicated that the DEGs were closely associated with cell migration and angiogenesis (Fig. 2D). Meanwhile, KEGG pathway analysis showed that DEGs were enriched in VEGF pathways and some pathways in cancer (Fig. 2E).
OCT4 induced angiogenesis and ovarian cancer cell metastasis via VEGFR2-LRPPRC signaling axis Through above Fig. 2D-E we inferred that OCT4 was associated with angiogenesis in ovarian cancer. Moreover, GSEA analysis of the RNA-sequencing showed that OCT4 knockdown signi cantly restrained vascular generation (Fig. 4A). To verify the assumption, we performed tube formation assay in human umbilical vein endothelial cells (HUVECs), which indicating that when HUVECs were cultured by ovarian cancer cells' supernatant from SKOV3ip1/OCT4-sh1, SKOV3ip1/OCT4-sh2, OVCA433/OCT4-sh1 and OVCA433/OCT4-sh2 cells, the tube formation was greatly inhibited by OCT4 silencing (Fig. 4B-C). As is well known, VEGFR2 is a crucial regulator and receptor in regulation of angiogenesis in VEGF pathway among a number of tumor vasculature-associated molecules. To probe into the interacting proteins of VEGFR2, immunoprecipitationi (IP) assay and mass spectrum (MS) analyses were performed, and 92 binding proteins were found (Supplemental table 2). Many of those proteins were cytoskeleton proteins, and nally we chose an interesting protein named LRPPRC for further sEtudy (Fig. 4D-E). And then, the interaction between VEGFR2 and LRPPRC was furtherly veri ed by Co-IP (Fig. 4F) and immuno uorescence (Fig. 4G).
Interestingly, via websites of Jaspar and NCBI we predicted that the transcription factor OCT4 might bind to upstream of LRPPRC promoter region. Firstly, we performed qRT-PCR assay and subsequently found that OCT4 knockdown decreased mRNA expression level of LRPPRC signi cantly (Fig. 5A). Next, we veri ed that the protein expression level of LRPPRC was also availably decreased by OCT4 silencing via immunoblotting and immuno uorescence ( Fig. 5B-C). Subsequently, we conducted ChIP assay to deeply explore the association between OCT4 and LRPPRC, and we concluded that OCT4 bound to speci c DNAbinding sites to induce LRPPRC transcription in the upstream region of transcription start site (TSS) (Fig.  5D). In the following study, we established LRPPRC silencing cell lines and observed that phosphorylation activation of VEGFR2 was inhibited by LRPPRC knockdown (Supplementary Fig. 1A). In general, we concluded that OCT4 induced angiogenesis and promoted cell migration and invasion via VEGFR2/LRPPRC pathway at the transcription level.

OCT4 activates vascular formation and promotes ovarian cancer progression and metastasis in vivo
In order to verify the above standpoints, subcutaneous and intraperitoneal nude mice models of ovarian cancer were generated. We investigated that xenograft tumor growth was restrained by OCT4 knockdown markedly, which was increased by the overexpression of LRPPRC signi cantly (Fig. 6A-F). After the mice in the control group of intraperitoneal xenograft model were sacri ced and dissected, we tested the association between OCT4 and LRPPRC via immuno uorescence, and veri ed that OCT4 silencing reduced LRPPRC expression in tumor tissues of ovaries (Fig. 6G). Then, we demonstrated that OCT4 silencing decreased the expression of vascular endothelial cell-speci c marker CD31 by immuno uorescence too (Fig. 6H). In our study, we constructed zebra sh models to detect angiogenesis in vivo. OCT4 morphlino suppressed zebra sh vascular formation, and the inhibitory action was turned back by overexpression of LRPPRC (Fig. 6I). Together, the ndings suggested that OCT4 acted as an oncogene and promoted angiogenesis and ovarian cancer metastasis in vivo.
The expression and association among OCT4, LRPPRC and VEGFR2 in human ovarian cancer specimens To detect the expression of LRPPRC, we performed IHC assay and observed that LRPPRC was substantially upregulated in human epithelial ovarian cancer samples, especially in metastatic lesions, compared with benign ovarian tumors (Fig. 7A). And then survival analysis demonstrated that high LRPPRC was correlated with poor OS and PFS in ovarian cancer patients (Fig. 7B).The positive ratio of LRPPRC in metastatic lesions of human ovarian cancer was 85.71%, which was obviously higher compared with the primary ovarian cancer specimens (66.84%), and both of them were higher than benign ovarian tumor tissues (38.56%, Fig. 7C). Following immuno uorescence assay indicated that OCT4 and angiogenesis biomarker CD31were greatly high expressed in ovarian cancer metastatic lesions (Fig. 7D). Additionally, via qRT-PCR we discovered that relative mRNA levels of angiogenesis and metastasis-related proteins such as VEGFA, AKT1 and N-cadherin were upregulated in OCT4 high expressed ovarian cancer tissues, and simultaneously E-cadherin was markedly reduced (Fig. 7E). Ultimately, we summarized the mechanism in this study and drew the novel pathway of OCT4/VEGFR2/LRPPRC in ovarian cancer (Fig. 7F).

Discussion
Our study clari ed a new molecular mechanism of metastasis in ovarian cancer. Brie y, initial immunohistochemistry and immuno uorescence analyses disclosed that OCT4 was signi cantly enhanced in ovarian cancer metastatic lesions; subsequently, OCT4 induced angiogenesis and ovarian cancer cell metastasis via VEGFR2/LRPPRC axis. Thus, our ndings highlight the role of OCT4/LRPPRC/VEGFR2 axis in metastasis of ovarian cancer, which supplied promising therapeutic targets for ovarian cancer.
OCT4, essential for the maintenance of stem cell phenotypes and pluripotent characters [15], has been veri ed to be indispensable for cancer cell stemness [16], and discovered to boost oncogenesis [17,18].
OCT4 was reported to be remarkably high expressed in lung adenocarcinoma [19], colorectal cancer [20], cervical cancer [21], ovarian cancer [22], and so on. Our nding indicated that OCT4 could activate angiogenesis and promote ovarian cancer metastasis by regulating LRPPRC/ VEGFR2 axis.
LRPPRC belongs to the PPR motif-containing proteins family, which can regulate transcription and the process of translation [23]. It was shown that gene mutation of LRPPRC led to Leigh syndrome French-Canadian [24]. Nowadays, more and more studies have demonstrated that LRPPRC dysregulation is associated with tumors. LRPPRC expression was increased in various tumor cell lines and cancer tissues, including colorectal cancer [25],gastric cancer[26], prostate cancer [27,28], and so on. Moreover, LRPPRC plays an important function in the progression of some cancers and predicts poor prognosis [29], and the restriction presents a promising potential molecular strategy for the tumor therapy [28]. Current studies about signal pathways of LRPPRC are limited, and LRPPRC has been seldom reported in ovarian cancer. Our study demonstrated that LRPPRC was high expressed in ovarian cancer, especially in the metastatic lesions, and LRPPRC could bind to VEGFR2 to activate it to promote angiogenesis and metastasis of ovarian cancer cells nally.
Angiogenesis participates in multiple processes of tumor behavior, including metabolism [30], metastasis processes [31], and cancer stem cell maintenance [32,33]. Vasculature is largely quiescent in the normal conditions [34], but an "angiogenic switch" is usually aroused in tumors by several angiogenic factors resulting in vascular formation [35]. Nowadays, antiangiogenic treatments have shown e cacy for some malignancies such as colorectal cancer [36,37], epithelial ovarian cancer [38,39] and non-small-cell lung cancer [40,41]. Interestingly, we proved that OCT4 knockout inhibited angiogenesis via inhibiting VEGFR2/LRPPRC pathway. Then, we infer that the OCT4/LRPPRC/VEFGFR2 axis may be an important therapeutic target in ovarian cancer, though it demands further clinical veri cation.

Conclusion
On the whole, our nding supplied a novel mechanism of action of metastasis in ovarian cancer, and provided a potential antiangiogenic strategy via targeting OCT4/LRPPRC/VEFGFR2 signaling pathway.

Tissue specimens
In our study, 153 benign ovarian tumor tissues, 187 primary lesions of epithelial ovarian cancer, and 154 metastatic specimens from greater omenta were collected after acquisition of the informed consent in Xinhua Hospital (Shanghai, China). Tissue samples were gained between 2008 and 2017. Overall survival (OS) was calculated as the interval from surgery initiation to death for whatever reason or nal time for follow-up. Progression-free survival (PFS) was measured from surgery initiation to disease relapse or progression.

Ovarian cancer organoids generation
Ovarian cancer organoids were gained from human high-grade serous ovarian carcinoma (HGSOC). Organoids generation and culture were performed as previously described [42]. Fresh cancer tissues were promptly carried to laboratory and dipped in Advanced Dulbecco's Modi ed Eagle's Medium (DMEM)/F12. Tissues were diced into approximately 2-3 mm sections and subsequently digested in 37°C for 1 hour.

Wound healing assay
Firstly, cells were seeded on 6-well plates with 9 × 10 5 cells/well as con uent monolayers. Subsequently, scratch wound healing assay was performed by pipette tips. After incubating for 48 h, we calculated the migrated area using microscopy.

Transwell assays of migration and invasion
Transwell migration and invasion assays were performed as previously described [43]. 5 × 10 4 cells were planted into the upper chamber plates and cultured with serum-free DMEM medium.
The lower chamber was added with DMEM containing10% FBS. Non-migrated or non-invasive cells were removed by cotton buds after 48 h, and the migrated or invasive cells were stained with crystal violet. Stained cells were counted in ve random elds per well under a light microscope (Nikon, Japan).

Immunoprecipitation
Pierce Crosslink Immunoprecipitation kit was utilized to perform immunoprecipitation (IP) following the manufacturer's protocol. In short, lysates of SKOV3ip1 ovarian cancer cells were generated via lysis buffer and then tested by BCA Protein Assay Kit (Beyotime, China). In the subsequent procedures, 1 mg total cell lysates were pre-cleared and blended with 10 µg antibody or IgG, and co-incubated with A/G agarose beads at 4℃ overnight. Finally, immunoblotting assay was carried out to examine the target proteins.

Mass spectrometry
Firstly, cell sediment was collected by centrifugation. And then, protein was extracted by 8 mol/L urea lysate, which was dissolved in 100 mmol/L ammonium bicarbonate. Through Bicinchoninic Acid (BCA) assay, protein concentration was determined and 300 µg protein was used for the following mass spectrometry (MS) assay. After processes of reduction reaction, puri cation and enzymatic hydrolysis, samples were desalted via the desalination column, and nally tested by mass spectrometer (Thermo Fisher, USA).

RNA sequencing
After total RNA was extracted by TRIzol (Thermo Fisher, USA), 1 mg RNA was used for subsequent RNA sequencing analysis. The method was detailed described as reported [44].
Immunohistochemistry assay Para n-embedded tissue was sliced into 3 µm thin sections for immunohistochemistry (IHC) analysis. Antibodies of OCT4 (ab181557, Abcam) and LRPPRC (ab97505, Abcam) were applied and subsequently incubated with corresponding secondary antibodies at 37°C for 1 h. Histochemistry score (H-score) and positive ratio were utilized to assess the expression level as described formerly [43].
Quantitative real time PCR Total RNA was centrifugated and extracted from ovarian cancer cells and tissues with TRIzol reagent (Invitrogen, US) in order to perform quantitative real time PCR (qRT-PCR). Primers in our study were exhibited in Supplemental Table 1.

Immuno uorescence assay
Ovarian cancer cells or tissues were xed with 4% paraformaldehyde at room temperature (RT) for 15 min and then incubated with 0.3% Triton X-100. After blocking of 5% goat serum (Life Technologies, US) at RT, cells or tissues were treated with appropriate amount of antibody overnight at 4 ℃. In the next step, primary antibody was cleared up by phosphate buffered saline (PBS) for 3 times, and then corresponding secondary antibody was added to the cells or tissues. 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies, US) was utilized for nuclear staining. Ultimately, the stained cells were visualized using a Leica SP5 confocal uorescence microscope.
Nude mice model BALB/c nude mice tumor associated experiments protocols were authorized and approved by Ethics Committee at Xinhua hospital. In this study, a total of 60 female mice (aged 4 weeks) were used. The models of subcutaneous and intraperitoneal ovarian cancer mice were generated by injection of 5 × 10 6 SKOV3ip1 cells. Subcutaneous tumor volume was regularly measured and calculated twice a week by vernier caliper. Intraperitoneal tumor volume of the ovarian cancer mode was observed by luminescence imaging techniques.

Zebra sh model
In our study, transgenic enhanced green uorescent protein (EGFP) expressing zebra sh model was used for assessing angiogenesis in vivo. OCT4 morpholino plasmid and/or LRPPRC cDNA were microinjected into 1-cell zebra sh embryos. After 24 hours post fertilization (Hpf), uorescence microscopy was used to detect the blood vessel formation in zebra sh model. For subsequent examination of VEGFA mRNA of zebra sh embryos, qRT-PCR assay was performed following the procedure above.

Statistical analysis
Data in our study were analyzed by GraphPad Prism 8.0 and SPSS 24.0 software. Student's t test was used for statistical analyses. The Kaplan-Meier survival analysis was performed to assess OS and PFS. p value less than 0.05 was considered as statistical signi cance.      was veri ed by Co-IP. (G) Immuno uorescence assay was applied to test the association between VEGFR2 and LRPPRC in ovarian cancer cells.