NANOG regulates epithelial–mesenchymal transition via AMPK/mTOR signalling pathway in ovarian cancer SKOV‐3 and A2780 cells

Abstract NANOG engages with tumour initiation and metastasis by regulating the epithelial–mesenchymal transition (EMT) in epithelial ovarian cancer (EOC). However, its role in association with pAMPKα, and its clinical significance in EOC have not been elucidated even though AMPK is known to degrade NANOG in various human cancers. Hence, we investigated the role of pAMPKα and its association with NANOG as potential prognostic biomarkers in EOC. Both NANOG and pAMPKα expression were significantly overexpressed in EOCs comparing nonadjacent normal epithelial tissues, benign tissues, and borderline tumours. NANOG overexpression was significantly associated with poor disease‐free survival (DFS) and overall survival (OS), whereas pAMPKα overexpression was associated with good DFS and OS. Importantly, multivariate analysis revealed that the combination of high NANOG and low pAMPKα expression was a poor independent prognostic factor for DFS and was associated with platinum resistance. In ovarian cancer cell lines, siRNA‐mediated NANOG knockdown diminished migration and invasion properties by regulating the EMT process via the AMPK/mTOR signalling pathway. Furthermore, treatment with AMPK activator suppressed expression of stemness factors such as NANOG, Oct4 and Sox2. Collectively, these findings established that the combination of high NANOG and low pAMPKα expression was associated with EOC progression and platinum resistance, suggesting a potential prognostic biomarker for clinical management in EOC patients.


| INTRODUC TI ON
Based on data from global cancer statistics (Global Cancer Incidence, Mortality and Prevalence [GLOBOCAN]), ovarian cancer is the 8th most common cancer among women globally, with an estimated 313,000 new cases and 207,000 deaths in 2020. 1 Despite improvements in treatment, such as target therapy or immunotherapy, 2 the prognosis of patients with epithelial ovarian cancer (EOC) remains poor due to a lack of effective clinical screening methods, resulting in diagnoses at advanced stages. 3 As such, most EOC patients have metastatic disease at the time of diagnosis. 4 Thus, it is crucial to investigate biomarkers for the early detection of EOC as well as their metastasis to reduce disease mortality and platinum resistance.
Epithelial-mesenchymal transition (EMT) is essential for the initiation of metastasis for cancer progression. Recent studies have suggested that ovarian cancer cells acquire mesenchymal traits and lose epithelial traits when they attain the ability to invade. 5 EMT-positive status has been correlated with poor progression free survival and overall survival. 6 Furthermore, in metastatic serous ovarian carcinoma effusions, several proteins related to EMT, including vimentin and ZEB1, have been found to be markers of a poor chemotherapy response. 7 Recent studies have highlighted a link between EMT and the properties of cancer stem cells (CSCs). 8 As a central stemnessassociated transcription factor, NANOG regulates the fundamental properties of CSCs, such as cell proliferation, cell cycle, selfrenewal, EMT, tumorigenicity, and chemoresistance. [9][10][11] In ovarian cancer, the androgen receptor contributes to the function of NANOG, which subsequently promotes ovarian CSC maintenance.
Samples of metastatic foci as well as ovarian cancer cell lines with features associated with metastasis have both been found to have high NANOG expression. 12 Additionally, in patients with colorectal cancer, breast cancer, and ovarian serous carcinoma, NANOG expression has been linked to poor prognosis. [13][14][15] As EMT and the metastatic cascade are highly energy-consuming, the balance between ATP consumption and production could be critical for the motile and invasive capacities of cancer cells. AMP-activated protein kinase (AMPK), known as a master regulator of energy homeostasis, is pivotal in cancer metastasis as well as cancer cell metabolism. 16,17 Numerous studies have shown that AMPK suppresses EMT in various cell types, such as tubular epithelial cells, 18,19 breast cancer cells, 20 lung adenocarcinoma cells 21 and bronchial epithelial cells. 22 Moreover, increased expression of phosphorylated AMPKα (pAMPKα) in solid tumours, including breast, lung, and gastric cancers, has been shown to be associated with both prognosis and tumour grade. [23][24][25] Given that AMPK is associated with cancer metastasis, we hypothesized that NANOG modulates AMPK signalling to regulate the EMT process and promote ovarian cancer metastasis.
In this study, we investigated the clinicopathological features and prognostic significance of NANOG and pAMPKα in ovarian cancer. Additionally, we identified a novel mechanism by which NANOG increases the metastatic potential of ovarian cancer cells by regulating the AMPK/mTOR pathway, which might foster new therapeutic strategies for the treatment of ovarian cancer.

| Tissue microarray and immunohistochemistry
TMA, which was constructed in previous studies, was used in this study. 27,28 The TMA blocks were cut to 5μm thickness with a rotary microtome. After sectioning, the TMA sections were deparaffinized with xylene and dehydrated in serially graded ethanol to distilled water. Then, antigen retrieval was performed by incubating TMA sections using a steam pressure cooker (Pascal; Dako, Carpinteria, CA) in heat-activated antigen retrieval buffer at pH 6.0 (Dako) for anti-NANOG and at pH 7.8 for anti-phospho AMPKα (Thr172). The sections were treated with 3% H 2 O 2 solution in methanol for 10 min to block the endogenous peroxidase activity. After rinsing the slides, they were stained with an anti-NANOG antibody (rabbit antibody, clone#4903S, 1:200; Cell signalling Technology, Inc., Danvers, MA) for 1 hour and anti-pAMPKα antibody (rabbit antibody, clone#2535, 1:52; Cell signalling Technology, Inc.) for 32 min at room temperature.
Subsequently, the antigen-antibody reactions were visualized by using Envision + Dual Link System-HRP (Dako) and DAB + (3, 3′-diaminobenzidine; Dako) for 10 min. The stained sections were dehydrated and counterstained with haematoxylin and mounted in Faramount aqueous mounting medium (Dako). Appropriate negative and positive controls were included.

| Evaluation of IHC staining
The stained TMA sections were scanned using a high-resolution optical scanner (NanoZoomer 2.0 HT; Hamamatsu Photonics K.K., Hamamatsu City, Japan) at a 20× objective magnification (0.5 μm resolution). The scanned sections were analysed with Visiopharm software, version 4.5.1.324 (Hørsholm, Denmark). Brown staining intensity was scored on a scale by intensity from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, and 3 = strong) and percentage of the cytoplasm-stained tumour cells (range, 0-100) was obtained by using a predefined optimized algorithm. The overall histoscore was calculated by multiplying the percentage of the positive cells and intensity score (score range: 0-300). 29

| Cell culture and reagents
The human ovarian cancer cell lines, SKOV-3 and A2780 were

| Boyden chamber assay
To examine cell invasion, 48-well micro chemotaxis chambers

| Real-time quantitative RT-PCR
Total RNA was isolated using the AccuPrep® Universal RNA Extraction Kit (Bioneer), and cDNA was synthesized using

| Statistical analysis
The Mann-Whitney test or Kruskal-Wallis test was performed for statistical analysis of pAMPKα and NANOG expression levels, as appropriate. The Kaplan-Meier method was used to analyse the overall survival (OS) and disease-free survival (DFS) curves by subgrouping NANOG as high or low expression group and pAMPK as low or high expression group by using the optimal cut-off point calculated by "MaxStat" package of R software. 30 In addition, the Cox proportional hazard model was used to calculate hazard ratios and confidence intervals (CIs) in both univariate and multivariate models. Statistical analyses were performed by using SPSS version 25.0 (SPSS Inc., Chicago, IL). Statistical significance was set at p < 0.05.  Figure 1A,B). In case of pAMPK, we observed abundant expression in cytoplasm, and significantly higher expression in EOCs comparing nonadjacent epithelial tissues (p < 0.001; Table 1, Figure 1A,B).

| pAMPK and NANOG protein expression in ovarian cancer
In addition, both NANOG and pAMPK were significantly associated with serous cell type (p = 0.038, p < 0.001; Table 1, Figure 1B, respectively). However, when looking at FIGO stage, NANOG was associated with advanced FIGO stage whereas pAMPK was significantly associated with early FIGO stage. Additionally, high NANOG expression was associated with advanced tumour grade (p < 0.001; Table 1).

| Prognostic significance of pAMPK and NANOG expression
Next, we evaluated the prognostic significance of NANOG and pAMPK expression in EOC patients via a Kaplan-Meier curve, which demonstrated that the patients with higher NANOG expression (NANOG high ) were significantly associated with poor DFS and OS compared to lower expression of NANOG (NANOG low ) in EOC patients (p < 0.001, p = 0.008; Figure 2A,B). Meanwhile, higher pAMPK (pAMPK high ) was significantly associated with better DFS and OS compared with lower pAMPK (pAMPK low ) expression in EOC patients (p = 0.006, p = 0.004; Figure 2A, Figure 2C). Most importantly, the combination of NANOG and pAMPK (NANOG high /pAMPK low ) showed a higher prognostic value for chemotherapy-resistance than single protein expression (p = 0.039; Figure 2C).

| NANOG induces ovarian cancer cell migration and invasion by inhibiting the AMPK/mTOR signalling pathway
Given that NANOG expression stimulates ovarian cancer cell invasion and migration, we subsequently explored the AMPK/mTOR signalling pathway, which plays an important role in tumour development and metastasis. In addition, AMPK has been observed to exhibit tumour suppressive-like activity in ovarian cancer cells. 34 To examine the effects of NANOG on AMPK signalling, we firstly analysed AMPK activation by detecting phospho-AMPKα (Thr 172 ).
In SKOV-3 and A2780 cells, suppression of NANOG expression resulted in decreased phosphorylation of mTOR and p70S6K, a key mTORC1 target, as well as increased phosphorylation of AMPK ( Figure 4A). These results demonstrate that NANOG knockdown activates the AMPK/mTOR signalling pathway.
To verify that the effects of NANOG on migration and invasion were mediated by AMPK in ovarian cancer cells, we used the AMPK inhibitor, compound C, in siNANOG-transfected SKOV-3 and

| AI C AR SUPPRE SS E S EMT AND E XPRE SS I ON OF S TEMNE SS -REL ATED G ENE S IN OVARIAN C AN CER CELL S
To further investigate whether AMPK activation is sufficient to suppress cell migration and invasion, and EMT progression in ovarian cancer cells, explored the effect of the AMPK activator AICAR.
Treatment with AICAR greatly increased the phosphorylation of AMPK, which was accompanied by decreased phosphorylation of mTOR and p70S6K ( Figure 5A). In addition, AICAR suppressed EMT by inhibiting the expression of mesenchymal marker, ZEB-1, vimentin and N-cadherin. Boyden chamber assays showed that AICAR-treated SKOV-3 and A2780 cells exhibited reduced migration ( Figure 5B) and invasion ( Figure 5C) compared with untreated cells. Collectively, these data indicate that AICAR may inhibit ovarian cancer cell invasion and migration by targeting EMT.
As AMPK has been shown to be involved in modulating NANOG stability and expression, 32,35 we also investigated whether AMPK regulates stemness-related gene expression in ovarian cancer cells.
As shown in Figure 5D, AICAR exhibited a significant decrease in mRNA levels of NANOG, Oct4 and Sox2, which is in good agreement with a previous report demonstrating the effects of AICAR on the self-renewal and differentiation of mES cells. 36 These data indicate that AMPK activation by AICAR may lead to decreased cancer stemness by regulating the expression of stemness-related genes such as NANOG, Oct4 and Sox2 in ovarian cancer cells.

| DISCUSS ION
The transcription factor NANOG plays a role in embryonic stem cell self-renewal and is essential for maintaining cancer stem cell properties. The deregulated and abnormal expression of NANOG appears to play a critical role in oncogenesis. 9 In the present study, we in-   subunit and its clinicopathological significance in ovarian cancer revealed that patients with high AMPK α2 expressions had a lower disease recurrence rate and better overall and disease-free survival rates. 39 In addition, low expression of AMPK β1 is consistent with the lower activity of AMPK in ovarian cancers that are metastatic, high-grade and advanced in stage. 34 In this study, we showed that low expression levels of pAMPK were significantly correlated with poor DFS and OS in EOC. Consistent with our results, many other studies have explored the prognostic value of AMPK in human cancers, including gastric cancer, colorectal cancer, lung cancer, renal cell carcinoma and hepatocarcinoma. 24,25,[40][41][42] EMT is critically involved in tumour invasion, metastasis and resistance to therapy. Therefore, the molecular players involved in this process represent attractive targets in oncology. As NANOG high /pAMPK low was revealed to be a more valuable predictive biomarker for DFS and chemotherapy response in EOC patients than single protein expression, we next focused our attention on the mechanism of EMT progression by NANOG and the role of AMPK in this process. Our data showed that siRNAmediated NANOG knockdown drastically inhibited cell migration and invasion as well as the EMT process by activating the AMPK/ mTOR signalling pathway in SKOV-3 and A2780 ovarian cancer cells. However, we were unable to elucidate the precise molecular mechanism of AMPK activation by NANOG knockdown in this study. One of the potential hypotheses to explain this mechanism is the effect of the expression of Twist1 and Bmi1, which are wellknown targets of NANOG, 43

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
Data available on request from the authors.

I N S TITUTI O N A L R E V I E W B OA R D S TATE M E NT
All biological samples were collected after obtaining informed consent from participants, following the guidelines of the institutional review board (IRB) of Gangnam Severance Hospital (IRB No. 3-2020-0377).