High neuropilin and tolloid‐like 1 expression associated with metastasis and poor survival in epithelial ovarian cancer via regulation of actin cytoskeleton

Abstract Abnormal expression of neuropilin and tolloid‐like 1 (NETO1) has been detected in some human carcinomas. However, the expression of NETO1 and the underlying mechanism in epithelial ovarian cancer (EOC) remain unknown. In this study, we found that a higher NETO1 expression in EOC tissue samples compared to normal ovarian tissue samples was significantly correlated with worse overall survival. Additionally, Cox regression analysis suggested that NETO 1 was independently associated with overall survival. NETO1 overexpression enhanced the EOC cells’ migration and invasion capability in vitro via regulation of actin cytoskeleton. Mechanistically, silencing NETO1 reduced the expression of β‐tubulin, F‐actin and KIF2A. In conclusion, our results demonstrated the critical role of NETO1 in EOC invasion, and therapies aimed at inhibiting its expression or activity might significantly control EOC growth, invasion and metastatic dissemination.

NETO1 (neuropilin and tolloid-like 1), a protein coding gene, encodes a transmembrane protein containing two extracellular CUB domains. Research has revealed that this gene serves as an ionotropic glutamate receptor and can encode a protein that is involved in spatial learning and memory by regulating the function of neuronal N-methyl-D-aspartate receptor complexes in the hippocampus. An important paralog of NETO1 is NETO2. Recent studies have shown that NETO2, which is primarily implicated in neuron-related processes, is expressed in diverse human tumour types. A five-gene hepatic signature including NETO2 may play a feasible role in the prediction of tumour growth and risk of death in patients with hepatocellular carcinoma (HCC), suggesting a useful molecular tool for therapeutic management of HCC patients. 6 A recent study identified 21 genes including NETO1 that could promote bowel metastases in EOC and evaluated their potential significance as molecular targets for treatment of advanced EOC patients. 7 Taken together, these findings imply that NETO1 might contribute to carcinogenesis, tumour progression and poor clinical outcomes. However, NETO1 expression in EOC patients and its relationship with clinicopathological features have not been evaluated till date. Moreover, mechanisms involving NETO1 that underly EOC progression remain to be elucidated.
Therefore, in this study, both traditional laboratory studies and bioinformatic analysis were performed to investigate the potential role of NETO1 in carcinogenesis, invasion and progression in EOC patients. Furthermore, we chose the regulation of actin cytoskeleton 8-10 as the possible signalling pathway that might be involved in the development of EOC via bioinformatics analysis. A potential molecular target may help in selecting therapeutic markers in EOC patients with malignant bowel obstruction as well as prolong the survival time and improve the quality of life of these patients.

| Patient tissue samples
All specimens were collected by specialized personnel from patient visited Gynecology Department and given informed consent, Affiliated Hospital of Nantong University between July 2005 and July 2010. A total of 13 cases of normal ovarian tissues, resected from double adnexal hysterectomy, and 98 cases of ovarian cancer tissues were gathered and detected in our study. There were 69 cases of serous carcinoma and 29 cases of other types for tumour classification, and 60 cases of stage I and 38 cases of stage II-IV regarding of FIGO stage in total 98 cases. Detailed clinicopathological materials including patients' age, tumour grade, 5-year survival outcome follow-up and some other information were shown in Table 1. The protocol of selecting patients' samples in our study was described as before. All patients were followed up from the time of being diagnosed till July 2019. The protocol of obtaining clinical samples was approved by the Ethics Committee of Affiliated Hospital of Nantong University.

| TMA construction and IHC analysis
The NETO1 protein expression was detected by using the tissue microarray (TMA) system to perform immunohistochemistry (IHC) analysis in 98 cases of EOC tissues. The method of TMA construction has been previously described. Immunostaining was performed using rabbit monoclonal antibody to NETO1 (1:200; Abcam, Cambridge, MA, USA) as the primary antibody and then incubated with the secondary antibody (SantaCruz, CA, USA).
Phosphate buffered saline severed as negative controls (NCs). Two independent pathologists evaluated and scored the NETO1 semiquantitative expression of each slide without knowing the clinical information of the case. We took the staining intensity and the percentage of positively staining cells into account for the scoring method, which demonstrated previously. The final immunostaining score was evaluated by the following criteria: staining intensity scores (no staining = 0, weak = 1, medium = 2, strong = 3) and positive staining ratio of NETO1 (ranged between 0 and 100).
The total score (staining intensity score × positive staining ratio) of NETO1 expression was divided into low expression and high expression groups. The X-tile software program was performed to set the cut-off point (99) for the NETO1 expression scores which was of statistically significance in terms of survival outcome. The expression scores were as below: low expression 0-99 and high expression 100-300 for NETO-1 in EOC tissues.
According to the manufacturer's instructions, cells were transfected with Lipofectamine ™ 3000 (3 μL/well; Invitrogen, Carlsbad, California, USA) and then added to the six-well plate for 4 hours After cultured in fresh complete medium for 24 hours, the interference efficiency of NETO1 gene was detected by Western blot. We used wound healing migration assay to evaluate migration ability. Cells were harvested in the logarithmic growth phase, adjusting cell concentration to 2.5 × 10 5 cells/mL, then seeded in a 24-well plate with 1 mL per well, and cultured at 37°C in a 5% CO 2 incubator.

| Invasion and migration assays
Cells transfected with shRNA-NC and shRNA-1, respectively, were cultured for 48 hours, and then, we used a 200 μL pipette tip to prepare a mechanical damage model. The damaged cell debris was washed gently by PBS, and after adding the fresh medium, the cells' reaction was observed under an inverted phase contrast microscope.

| GSEA enrichment analysis
Gene set enrichment analysis (GSEA), a calculation method that could estimate whether a list of previous defined genes shows concordant differences with statistically significant between two biological processes. 11 This study employed the GSEA to illuminate the significant difference in survival rates observed between the low and high NETO1 groups after initially generating a sequential list of all genes according to their relationship among NETO1 expressions.
For each analysis, the gene set permutations were performed 1000 times. The phenotype label was identified in the level of the NETO1 expression. In order to sort out the pathways enriched in each phenotype, the Normalized Enrichment Score (NES) and the nominal Pvalue were utilized.

| Survival outcome and multivariate analysis
The overexpression of NETO1 was associated with advanced cancer biology, indicated by metastasis, FIGO stage and serum CA125 level.
Kaplan-Meier curve analysis showed that EOC patients with a higher NETO1 expression and advanced stage had a poor overall survival (log rank, P < 0.001, Figure 1B,C).
To confirm that NETO1 overexpression may serve as an independent predictive factors for poor outcome in EOC patients,  Figure 1D,

| Metastasis-related cancer biology including cell migration and invasion
To investigate whether the malignant phenotype of EOC was related to abnormal NETO1 expression, Western blot analysis was con-   These results indicate that NETO1 expression silencing decreased the migration and invasion capability of EOC cells.

| Gene set enrichment analysis
To uncover the regulatory mechanisms underlying the functional effects of NETO1 and to identify the signalling pathways differentially activated in EOC, GSEA was performed by comparing high and low NETO1 expression data sets. In the enrichment of

| Regulation of actin cytoskeleton
On the basis of evidence from GSEA, we chose regulation of actin cytoskeleton as a possible signalling pathway that might be involved in development of EOC. Immunofluorescence microscopy indicated that cells expressing NETO1-shRNA had reduced levels of β-tubulin and F-actin expression, a key signalling protein in the regulation of actin cytoskeleton ( Figure 4A). Furthermore, Western blot analysis revealed that cells expressing NETO1-shRNA exhibited reduced KIF2A expression levels, which is a motor protein engaged in transporting cargo proteins along the microtubules (MTs; Figure 4B), suggesting that NETO1 is an upstream regulatory element for KIF2A and affects tumour biology through the regulation of actin cytoskeleton in EOC.   Our recent study established that KIF2A, a protein suppressed by miR-206, is relevant in the poor prognosis of ovarian cancer. 18 Notably, KIF2A is dysregulated in several other types of human cancers and functions as an oncogene in oral squamous cell carcinoma, 19 breast cancer, 20 human glioma, 21

ACK N OWLED G EM ENT
This study was funded by the National Natural Science Foundation of China (No. 81802606).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. The public data that support the findings of this study are openly available in GEO database at (https://www.ncbi.nlm.nih.gov/). 12