Metformin Exhibits an Attractive Antineoplastic Effect on Human Endometrial Cancer by Regulating the Hippo Signaling Pathway

Metformin, the first-line oral antidiabetic medicine, has shown great antineoplastic potential in various cancer types, despite an unclear mechanism. This study aimed to elucidate the possible mechanism of metformin as a chemotherapy agent with less reproductive and genetic toxicity in human endometrial cancer. The type I endometrial carcinoma cell lines Ishikawa and RL95-2 were treated with metformin. Cell functions, such as proliferation, migration, and invasion, were analyzed. Flow cytometry was performed for cell cycle and apoptosis analyses. Simultaneously, RT-qPCR and western blotting were performed to explore the possible mechanism. Moreover, YAP1 knockout Ishikawa cells were established via lentivirus to demonstrate the underlying mechanism. The results showed that metformin mediated Ishikawa and RL95-2 cell growth inhibition in a dose- and time-dependent manner. The IC50 values of metformin in Ishikawa and RL95-2 cells were 10 mM and 8 mM, respectively. The migration and invasion abilities were also inhibited in the metformin-treated group using wound healing assays and transwell migration and invasion assays, and Ishikawa and RL95-2 cells were arrested in the G1 or G2 phase, respectively. Moreover, the cell proportions of cells in both early and late apoptosis stages were dramatically elevated when treated with metformin, as was the ratio of Bax/Bcl-2 expression. Additionally, the expression levels of YAP1 mRNA and protein in the treatment group were much lower than those in the control group. The cellular behaviors of YAP1 knockout Ishikawa cells were similar to those in the metformin-treated group. Our results demonstrated that it is an attractive alternative to cytotoxic chemotherapy in human endometrial cancer, and YAP of the Hippo pathway may be a potential molecular target. This study provides novel ideas for the adjuvant therapy of endometrial cancer patients, especially for women with strong fertility desires and demands.


Introduction
Endometrial cancer is the most frequently occurring gynecologic malignancy in Western countries. It has been reported that since 2008, the incidence of endometrial cancer has increased by 21% in the United States, as well as doubling the death rate over the past two decades [1]. Although the utilization of nanotechnology has greatly impacted cancer therapeutics [2][3][4][5], chemotherapy is still a common adjuvant therapy for advanced or recurrent endometrial cancer; however, the devastating blow to the female reproductive system makes traditional platinum-based chemotherapy unsuitable for women who wish to preserve fertility [6]. erefore, the development of new chemotherapeutic agents that have comparable efficacy and fewer adverse effects on the reproductive system for endometrial cancer patients of reproductive age is needed. e Hippo signaling pathway is an evolutionarily conserved signaling transduction and regulator involved in cell differentiation, tissue regeneration, organ development, and immune modulation [7]. e core of this signaling cascade comprises kinases such as large tumor suppressor 1/2 (LATS1/ 2) and the transcriptional coactivator Yes-associated protein (YAP) [8]. Phosphorylation of YAP activated by LATS kinases results in their cytoplasmic retention and degradation, which causes the failure of their binding with transcriptional enhanced associate domains (TEADs) and other potential transcription factors in the nucleus to repress gene transcription [9,10]. Recently, an increasing number of studies on human carcinomas have validated the significant role of the Hippo pathway in tumorigenesis and tumor progression [11]. YAP is also upregulated and closely associated with the degree of malignancy and prognosis in several gynecologic cancers, including cervical [12], ovarian [13], and endometrial cancer [14].
us, cumulative evidence has demonstrated YAP as a possible new molecular target for cancer therapy [15][16][17], and it is practical to seek an alternative Hippo-targeted agent with limited reproductive and genetic toxicity for endometrial cancer.
Metformin, the most widely used antidiabetic drug [18], has been proposed to have antineoplastic properties against certain types of cancers, including gynecological cancers [19,20]. Metformin has been reported to attenuate the stemness of glioma cells [21] and suppress cell proliferation and invasion in non-small cell lung cancer [22] by downregulating the expression of YAP. Considering the high expression of YAP in endometrial cancer, it is reasonable to speculate the possible link between the inhibitory effect of metformin on endometrial cancer and the Hippo signaling pathway. Moreover, metformin has also exhibited reproductive endocrine modulatory properties [23,24], which makes metformin an attractive alternative to cytotoxic chemotherapy. e present study aims to explore the antineoplastic effect of metformin on endometrial cancer in vitro and the role of the Hippo signaling pathway in the process.

Cell Viability Assay.
Cell function assays included cell viability assays, flow cytometry analysis, wound healing assays, transwell migration assays, and in vitro transwell invasion assays. Cell viability was evaluated by CCK-8 assay using a CCK-8 kit purchased from MCE, China, following the manufacturer's instructions. Briefly, cells were incubated with various concentrations of metformin for different times. en, 10 μL CCK-8 reagent was added to the medium 2 h before OD450 measurement using a spectrophotometer (BioTek, United States).

Flow Cytometry Assay.
e cell cycle was analyzed using flow cytometry. Cell pellets were resuspended in precooled 90% ethanol and then incubated at −20°C overnight. After washing and incubating in a water bath, the cells were stained with propidium iodide (PI, BD Biosciences, United States) for 20 min in the dark before flow cytometry analysis. Dual staining was performed for the apoptosis assay using an Annexin V-FITC/PI kit (Cat. No. 556547, BD Biosciences, United States). Cells stained with FITCconjugated Annexin V and PI were measured by flow cytometry.

Wound Healing Assay.
Well-spread cells in 24-well plates were scratched by a pipette tip after FBS starvation for 12 h and then observed every 24 h using an inverted microscope (Axio Observer A1, Carl Zeiss, Germany). e relative wound areas were quantified and calculated by ImageJ (NIH, United States).

Transwell Migration and Invasion
Assay. For the transwell migration and invasion assays, 10,000/mL cells in 100 μL FBS-free medium were seeded in the top chambers of transwells (for the migration assay, Corning, United States) and Matrigel-coated transwells (for the invasion assay, Corning, United States), and the medium on the bottom was supplemented with 20% FBS. After 48 h of culture, cells in the chambers were fixed with 4% paraformaldehyde, stained with crystal violet (Servicebio, China), and then counted under a 20× microscope (Axio Observer A1, Carl Zeiss, Germany).

Statistical
Analysis. Statistical analyses were performed by using SPSS version 26.0 (IBM, United States), and data visualization was carried out using GraphPad Prism 8.0 (GraphPad Software Inc., United States). All data are presented as the mean ± standard deviation (SD). Student's independent t-test was used for comparisons between two groups, and analysis of variance (ANOVA) was used for comparisons among three groups. Two-tailed hypothesis tests were performed. P < 0.05 was considered significant. Each experiment was performed in triplicate and repeated at least three times independently.

Metformin Inhibited Cell Proliferation and Induced Cell
Cycle Arrest in Endometrial Carcinoma Cells. Ishikawa and RL95-2 cells were incubated with metformin at different concentrations (0, 5, 10, 20, and 40 mM for Ishikawa cells and 0, 5, 10, and 20 mM for RL95-2 cells), and the cell proliferation ability was detected using CCK-8 assays. As shown in Figure 1(a), with the prolongation of the concentration of metformin treatment, the relative OD value of cells decreased sharply, which indicated that cell proliferation inhibition of Ishikawa was dependent on the dose of metformin exposure. e IC50 of metformin in Ishikawa cells was calculated, and the estimated value was 10 mM. en, Ishikawa cells were treated with 0, 10, and 20 mM metformin for various times (0, 24, 48, and 72 h), and significant time-dependent inhibition was demonstrated ( Figure 1(b)). A similar phenomenon can also be observed in RL95-2 (Figures 1(c) and 1(d)), and the IC50 value of metformin in RL95-2 cells was 8 mM. ese results indicated that metformin also mediated RL95-2 cell growth inhibition in a dose-and time-dependent manner. Based on the results of the CCK-8 assays, 48 h was chosen as the treatment time of the agent. To investigate the effect of metformin on endometrial carcinoma cells, the cell cycle was analyzed by flow cytometry. As shown in Figure 1(e), compared with the control group (Figure 1(g)), the proportion of Ishikawa cells in the G1 phase increased significantly, and the ratio of cells in the S phase decreased in the 10 mM (Figure 1(h)) and 20 mM (Figure 1(i)) metformin-treated group. is result demonstrated that metformin exposure resulted in cell cycle arrest at the G1 phase in Ishikawa cells. Similarly (Figure 1(f )), metformin was found to be capable of arresting the cell cycle at the G2 phase in PL95-2 cells (Figure 1(k)) in comparison with the nontreated group ( Figure 1(j)). To further confirm the flow cytometry results, the protein expression levels of Bcl-2 and Bax were tested using western blotting. e ratio of Bcl-2/Bax decreased in Ishikawa cells treated with metformin (Figures 2(e) and 2(f )), which indicated that the mitochondrial apoptosis pathway may be involved in apoptosis of Ishikawa cells induced by metformin.

Metformin Inhibited Cell Migration and Invasion in
Endometrial Carcinoma Cells. Ishikawa and RL95-2 cells were treated with metformin (10 and 20 mM for Ishikawa cells, 8 mM for RL95-2 cells) to investigate cell migration and invasion. Since confluence growth was absent in RL95-2 cells, the wound healing assay was only performed in Ishikawa cells. e relative areas between the scratches in the control group were positively correlated with exposure time, and the wound was almost healed after 48 h of treatment. In the metformin treatment group, the speed of wound healing was significantly slower, especially with a higher concentration of metformin, which indicated an inhibitory effect of metformin on Ishikawa cell migration (Figures 3(a) and  3(b)). e results of the transwell migration assay have also corroborated the inhibitory effect of metformin on Ishikawa and RL95-2 cell migration. e number of adherent Ishikawa cells in the control group was 111.1 ± 26.1 at 100× magnification, which was higher than that in the 10 mM at 100× magnification), the number of penetrating cells was much lower than that in the 10 mM metformin group (55.7 ± 33.3) and the 20 mM metformin group (20.6 ± 14.0) (P < 0.01) (Figures 3(g) and 3(h)). Similar results were also observed in the RL95-2 cells (Figures 3(g) and 3(j)).
e Hippo signaling pathway may be an antitumor target of metformin in endometrial carcinoma cells.
Ishikawa cells were treated with various concentrations of metformin (0, 10, and 20 mM) for 48 h to explore the possible mechanism of the antitumor effect of metformin. e mRNA and protein expression of YAP1 and LATS in Ishikawa cells were detected using qRT-PCR and western blotting.
e relative protein level of p-YAP1/YAP1 increased obviously in the metformin-treated group (Figures 4(a)-4(b)). Additionally, a dramatic decrease in the protein expression of LATS was observed in the treatment group (Figures 4(c) and 4(d)). Moreover, the YAP1 mRNA level showed a consistent trend with the protein level (Figure 4(e)).
ese results indicated that metformin can activate the Hippo signaling pathway in Ishikawa cells. e efficiency of YAP1 knockout in Ishikawa cells was confirmed using qRT-PCR and western blotting, and our results showed a dramatic decrease in the mRNA and protein expression of YAP1 in the YAP1-KO group compared with the BC and NC groups (Figures 4(f )-4(h)). e cell proliferation ability of YAP1-KO Ishikawa cells was investigated using CCK-8 assays, and the lack of YAP1 inhibited the cell proliferation of Ishikawa cells (Figure 4(i)). Additionally, a transwell invasion assay was performed to detect the effect of YAP1 deletion on in vitro invasion in Ishikawa cells, and the results demonstrated the obvious inhibition of invasion in YAP1-KO Ishikawa cells (Figures 4(j) and 4(k)). Equally, the knockout of YAP1 in Ishikawa cells also sharply increased the percentage of apoptosis in both the early and late apoptosis stages (Figures 4(l) and 4(m)).

Discussion
In the current study, we found that metformin was able to inhibit cell proliferation, migration, and invasion, as well as

Con
Met-8 mM   Journal of Oncology induce cell cycle arrest and apoptosis in human endometrial cancer cells. Moreover, YAP, the core of the Hippo signaling pathway, might be a potential target for the antineoplastic therapy with metformin in endometrial cancer ( Figure 5). First, Ishikawa and RL95-2 cells were treated with various concentrations of metformin at different times and analyzed by CCK-8 assay to seek a suitable treatment concentration and time. Cell cycle arrest in G1 and G2 phases can be observed by flow cytometry, and metformin is likely to interfere with the process of DNA replication. e results of Annexin V/PI dual staining flow cytometry also proved that metformin induced both early and late stages of apoptosis, which was further reinforced by western blotting. Moreover, the decrease in Bcl-2/Bax in the metformintreated group suggested the possible participation of the mitochondrial pathway. In addition, more cell functions were investigated through wound healing tests and transwell analysis, and significant inhibition of cell migration and invasion was observed. e above results indicated the significant suppression effects of metformin on endometrial cancer cells in vitro.
Metformin has shown great potential for antidiabetic, antiphlogistic, antilipemic, and antineoplastic properties. It has been reported that metformin reduces the incidence and death rate and improves the prognosis of multiple malignancies, including hepatocellular [25], pancreatic [26], ovarian [19], and cervical carcinoma [20]. However, the possible mechanism remains unclear. Some studies reported that metformin activated the AMP-activated protein kinase (AMPK) signaling pathway [27], inhibited the expression of the proto-oncogene human epidermal growth factor receptor 2 (HER-2) [28], and resulted in cell cycle arrest and proliferation inhibition [29]. In other studies, metformin has also been proposed to downregulate YAP expression to attenuate cell proliferation and invasion in non-small cell lung cancer [22]. Dysfunction of the Hippo signaling pathway is widely proven to promote tumorigenesis [30], and YAP, as the heart of the Hippo signaling pathway, acts as a coactivator to activate cell proliferation genes, such as connective tissue growth factor (CTGF) and brain-derived neurotrophic factor (BDNF) [9]. In our study, YAP was chosen as the target and knocked out in Ishikawa cells using the CRISPR/Cas9 system, which is an RNA-guided genome editing tool consisting of a Cas9 nuclease and a single-guide RNA (sgRNA). Similar tumor suppression induced by metformin can be observed, which supports the hypotheses that the Hippo signaling pathway is likely to participate in the regulation of cell death and the fate of endometrial cancer cells. It has been reported that YAP/TAZ, the transcriptional regulators of the Hippo pathway, are highly expressed in various cancers, and the elevation of YAP/TAZ levels in endometrial cancer cell lines has been proven to increase cell proliferation, migration, and invasion [31][32][33]. Moreover, TAZ/YAP was associated with the PI3K/AKT pathway in endometrial cancer, in which the activation of the Hippo pathways reduced the activation of the PI3K/AKT pathway, affecting multiple physiological processes [31,34]. In addition, YAP can interact with the promoter of IL-6, increasing the transcription level of IL-6, which is significant for the tumorigenesis of endometrial cancer cell lines [32]. In conclusion, the Hippo pathway is a new molecular mechanism for the development of endometrial cancer, providing novel insight into therapeutic strategies targeting the Hippo pathway, in addition to traditional therapeutics. e Ishikawa and RL95-2 cell lines both originated from type I endometrial cancers, which are hormone-dependent and caused by cumulated estrogen with the lack of the antagonism of progesterone. Type I endometrial cancer is usually well-differentiated adenocarcinoma, normally accompanied by obesity, diabetes, and infertility, and secondary to atypical hyperplasia of the endometrium [35]. Interestingly, type 2 diabetes mellitus (T2DM) is also known to be a key risk factor for endometrial cancer [36], and a meta-analysis involving 21 cohort studies showed an obvious positive relationship between a preexisting history of diabetes mellitus and the incidence of endometrial cancer,

Con
Met-8 mM  Journal of Oncology despite no significant risk of mortality [37]. Our study confirmed that the first-line antidiabetic metformin for T2DM had potential antineoplastic properties in endometrial cancer, which was also proven in another study [38]. In addition, several studies have emphasized the attentional link between T2DM and endometrial cancer risk and mortality, mainly through obesity and chronic inflammation [39]. Our findings shed light on the other possible connection between these two highly frequent diseases. Drug safety is always the focal concern of health care providers. Metformin has been proposed to improve the clinical outcomes of fertility in females suffering from polycystic ovary syndrome (PCOS) with insulin resistance [40]. Compared with traditional chemotherapy agents such as cisplatin, metformin showed less reproductive and genetic toxicity [23]. However, it should be noted that the concentrations of metformin used in our in vitro studies, with an IC50 of 8 to 10 mM, were much higher than those in animals, which were between 40 and 70 μM of the plasma concentration in veins after the administration of a therapeutic dose [41]. A similar study also revealed that metformin at an antidiabetic dose still exhibited an antineoplastic effect on cancer cells [38], but it was only a preclinical study and the conclusion still needs to be discussed and confirmed. erefore, a safe and effective dose of metformin must be explored in a well-established endometrial cancer animal model in the future.
ere were still limitations in this study. We only detected the expression of a few proteins and mRNAs involved in apoptosis and the Hippo pathway, and the exploration of the mechanism was not comprehensive and deep enough. In addition, our conclusions were based on in vitro cell experiments, and more animal studies are needed to reinforce our current results and conclusions. Moreover, the functional routes of metformin in endometrial cancer are complicated and cross-linked, and the Hippo signaling pathway may not be unique. e exact signaling pathway network remains to be explored. e lack of comparison of metformin and traditional antitumor drugs such as paclitaxel can be another limitation. Whether metformin can achieve a similar antitumor effect as other drugs is still unclear. More mechanistic studies with a better design are needed to investigate the anticancer effect of metformin on endometrial cancer in the future.

Conclusions
In conclusion, metformin treatment in human type I endometrial carcinoma Ishikawa cells and RL95-2 cells can inhibit proliferation, migration, and invasion, block the cell cycle, and promote apoptosis, demonstrating that metformin is an attractive alternative to cytotoxic chemotherapy in human endometrial cancer and that YAP of the Hippo pathway may be a potential molecular target.
is study provides novel ideas for the adjuvant therapy of endometrial cancer patients, especially for women with strong fertility desires and demands.

Data Availability
All data generated or analyzed during this study are included in this published article.  Journal of Oncology 9