KLF2 transcription suppresses endometrial cancer cell proliferation, invasion, and migration through the inhibition of NPM1

Abstract Endometrial cancer (EC) is the most common gynaecologic malignancy. This study was to explore the role of kruppel-like factor 2 (KLF2) in EC cell behaviours. The expression of KLF2 in EC and its correlation with NPM1 were first predicted on the database. Levels of KLF2 and nucleophosmin 1 (NPM1) in EC cell lines were then determined. After transfection of the overexpression vector of KLF2 or NPM1, cell proliferation, invasion, and migration were evaluated. The binding relationship between KLF2 and the NPM1 promoter was analysed. KLF2 was downregulated while NPM1 was upregulated in EC cells. KLF2 overexpression reduced the proliferation potential of EC cells and the number of invaded and migrated cells. KLF2 was enriched in the NPM1 promoter and inhibited NPM1 transcriptional level. NPM1 overexpression neutralised the effects of KLF2 overexpression on suppressing EC cell growth. Collectively, KLF2 was decreased in EC cells and KLF2 overexpression increased the binding to the NPM1 promoter to inhibit NPM1 transcription, thus suppressing EC cell growth. IMPACT STATEMENT What is already known on this subject? Endometrial cancer (EC) sees a significant increase in prevalence globally with poor survival rates. KLF2 has been recognised as a tumour suppressor in a variety of malignancies. NPM1 can regulate EC cell viability and apoptosis. What do the results of this study add? This study highlighted that KLF2 produces an anti-tumour effect in EC and KLF2 can regulate NPM1 expression in EC and thereafter affect the proliferative, migratory, and invasive abilities of EC cells. What are the implications of these findings for clinical practice and/or further research? KLF2 overexpression suppresses NPM1 transcription and inhibits EC cell growth. Targeting KLF2 overexpression may be useful for EC treatment in clinical practice.


Introduction
Owing to an ageing population, increasing incidence of obesity, and decreasing rates of benign hysterectomy, endometrial cancer (EC), gynaecological cancer, is experiencing a significant increase in prevalence globally (Crosbie et al. 2022).In 2020, approximately 417,367 women were diagnosed with EC around the globe and the number of annual cases is estimated to reach 122,000 by 2030 in the United States alone (Makker et al. 2021).The 5-year survival rates of EC patients at stages III and IV are 47-58% and 15-17%, respectively (Passarello et al. 2019).Although EC has profound impacts on postmenopausal women, the lifetime risk of EC for women aged 35 can reach 22-50% (Braun et al. 2016, Faria et al. 2019).EC is usually treated with surgery, and the recommendations for adjuvant treatment are few and controversial, not to mention the challenges of comorbidity and toxicity (Brooks et al. 2019).A large multicentre series demonstrated that the predictive score of nodal involvement has a diagnostic value of 55.7% in EC patients (Capozzi et al. 2022).Another study revealed that aberrant telomere length in circulating cell-free DNA can be used as a possible blood biomarker with high diagnostic performance in EC (Benati et al. 2020).The targeted therapy is gaining increasing academic interest recently in EC treatment and has shown promising effects on EC cell proliferation and migration.A further understanding of the molecular targets is critical to the selection of optimal treatment strategy (McDonald andBender 2019, Yen et al. 2020).
Kruppel-like factor 2 (KLF2) is a transcription factor and an important regulator of cell activation, differentiation, and migration (Wittner and Schuh 2021).KLF2 plays a pivotal role in the maintenance of endothelial barrier integrity and prevention of gap formation between endothelial cells and decreased KLF2 expression induces endothelial cell injury in diabetic mice (Rane et al. 2019).Moreover, KLF2 has been recognised as a tumour suppressor in a variety of malignancies such as ovarian cancer, hepatocellular carcinoma, and clear cell renal cell carcinoma (Wang et al. 2019b, Li et al. 2020, Lu et al. 2021).A recent study has pointed out that KLF2 is weakly expressed in EC (Mieszczanski et al. 2022).However, it is unknown if KLF2 produces an anti-tumour effect in EC.
Nucleophosmin 1 (NPM1) is a critical cellular protein whose function is required for normal cellular biology in cancers (Box et al. 2016).NPM1 has high expression levels in EC and a positive association with EC clinical stages and histological grades (Zhou et al. 2018).The knockout of NPM1 can reverse the effect of miR-30c inhibition on restoring EC cell viability and proliferation and reducing EC cell apoptosis (Shen et al. 2022).Intriguingly, KLF2 can suppress gene expression as a transcription factor, while NPM1 can be manipulated by transcription factors (Yung 2004, Li et al. 2021).Therefore, there is a possible interaction between KLF2 and NPM1.We hypothesise that KLF2 can regulate NPM1 expression in EC and thereafter affect the proliferative, migratory, and invasive abilities of EC cells.This article aims to probe into the function of KLF2 on EC cells and the molecular mechanism, with the expectation of offering novel theoretical references for EC management.

Cell culture
EC cell lines (HEC-1-A, Ishikawa, and KLE) and endometrial epithelial cells (EECs) were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (HyClone, Logan, UT, USA) and RPMI-1640 medium (HyClone) with 10% foetal bovine serum (Gibco), respectively at 37 � C with 5% CO 2 .Registration is not applicable.Since this was a cell-based study, no ethical approval was needed based on local regulations on medical science research.

MTT assay
EC cells were digested with trypsin to prepare cell suspensions.About 3000 cells per well were added to 96-well plates and incubated with 100 lL of culture medium.After cell transfection, cell proliferation was recorded at 0, 24, 48, and 72 h, respectively.At each time point, 20 lL (5 mg/mL) of preconfigured MTT reagent was added and incubated at 37 � C for 2 h at a constant temperature.The cell culture medium was removed, and cells were incubated with 150 lL of dimethyl sulfoxide in the dark with low-speed shaking.The absorbance value at 490 nm was then read using a microplate reader (BioTek, Winooski, VT, USA).

Colony formation assay
Colony formation assay was used to detect the role of KLF2 and NPM1 in EC.Briefly, 500 EC cells were seeded in 6-well plates and cultured in a medium containing 10% foetal bovine serum for approximately 2 weeks.Then, when the colonies were visible, the supernatant was discarded, washed carefully with PBS solution three times, and then the colonies were fixed with methanol and stained with crystal violet for 15 min.The stained colonies were rinsed with running water, air-dried, and counted under a microscope.Only colonies containing more than 50 cells were counted to determine the colony-forming capacity.

Transwell assays
For migration assays, cells (1 � 10 5 ) containing serum-free medium were placed in the upper chamber of a transwell chamber (8 lm pore size; Co-star, Cambridge, MA, USA).For invasion assays, cells (1 � 10 5 ) containing serum-free medium were cultured in the upper chamber containing Matrigel (BD Biosciences, Shanghai, China).The lower chamber was added with 500 lL of medium containing 10% foetal bovine serum.After 24 h incubation, the upper chamber was removed, washed three times with PBS solution, fixed in methanol for 15 min, and stained with 0.1% crystal violet for 20 min.Cells on the upper surface were wiped, and those infiltrating the submembrane surface were counted under light microscopy (Nikon, Tokyo, Japan).

Chromatin immunoprecipitation assay
ChIP was performed as previously described (Li 2021).Briefly, EC cells were fixed in paraformaldehyde for 10 min and then cut into pieces using an ultrasonic disruptor.The supernatant was collected by centrifugation (4 � C, 12,000 g, 10 min), and anti-IgG antibody (ab172730, Abcam) was added as NC, and anti-KLF2 antibody (PA5-40591, ThermoFisher) was added as the target antibody.After mixing, cells were incubated overnight at 4 � C. Protein A-Agarose/Salmon Sperm DNA precipitated the DNA-protein complex, and the pellet was centrifuged at 12,000 g for 5 min.The supernatant was discarded and the non-specific complex was washed and incubated overnight at 65 � C for de-crosslinking, and the DNA fragment was purified by phenol/chloroform extraction.The NPM1 promoter primers are shown in Table 1.

Dual-luciferase assay
The binding site wild or mutated recombinant luciferase reporter vector (NPM1 WT/NPM1 MUT) was co-transfected with KLF2/NC into EC cells.After 48 h of transfection, cells were collected and lysed.The luciferase activity was then measured using a dual luciferase assay system (Promega, Madison, WI, USA) and a luciferase assay kit (K801-200, Biovision, San Francisco, CA, USA).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from cells using TRIzol kits (Invitrogen) for RT-qPCR.cDNA was obtained using the Promega GoScript RT System (Promega), and the expression of KLF2 or NPM1 was detected on a Light Cycle 480 realtime qPCR instrument using the SYBR Green PCR Kit (Takara).The relative expression of genes was normalised to that of GAPDH and analysed using the 2 À DDCt method (Schmittgen and Livak 2008).All primers are listed in Table 1.

Western blot
Total protein was extracted from cancer cells using RIPA buffer (Kagen Biotechnology, Nanjing, China).The 10% SDS-PAGE was prepared for protein separation, after which proteins were transferred to PVDF membranes.The PVDF membranes were immersed in the sealing solution and shaken for 1 h at room temperature.The primary antibodies KLF2 (1:2000, ab236507, Abcam), NPM1 (1:1000, ab10530, Abcam), and b-actin (1:1000, ab8226, Abcam) were added into the membranes for overnight incubation at 4 � C.After washing with TBST (3 � 10 min) the next day, the membranes were probed with secondary antibody IgG (1:2000, ab205719, Abcam) for 1 h at room temperature.Finally, the bands were visualised with the help of an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA).

Statistical analysis
Data were statistically analysed and graphics were plotted using GraphPad Prism 8.01 (GraphPad Software, San Diego, CA, USA) and SPSS 21.0 (IBM Corp, Armonk, NY, USA).The data were verified as normally distributed through the Kolmogorov-SmiRnov test.Mean ± standard deviation was used to describe data.One-way or two-way analysis of variance (ANOVA) was used for comparisons of data between groups, with Tukey's test as a post hoc test.Statistical significance was signified by the difference of p < 0.05.

KLF2 was lowly-expressed in EC cells
The biological function of KLF2 in EC is unknown.We predicted on three databases that KLF2 was lowly expressed in EC (P < 0.05, Supplementary Figure S1(A-C)), and the OncoLnc database predicted a poorer prognosis for EC patients with low KLF2 expression (P ¼ 0.02, Figure S1D).To detect the KLF2 expression in EC, we cultured EC cells (HEC-1-A, Ishikawa, and KLE) and EECs and found significantly lower KLF2 expression in EC cells than EECs (Figure 1(A  and B)).

Overexpression of KLF2 inhibits EC cell proliferation
To investigate the role of KLF2 in cancer cells, we transfected KLF2 into Ishikawa (low relative KLF2 expression), which successfully upregulated intracellular KLF2 expression, and transfected si-KLF2 into HEC-1-A (high relative KLF2 expression), which decreased intracellular KLF2 expression (Figure 1(C and  D)), and selected transfected si-KLF2#2 with better efficiency for subsequent detection.Our results revealed that the proliferation in the KLF2 group was notably reduced and the number of cell colonies decreased compared with the NC group, while cell proliferation and colony number were substantially increased in the si-KLF2 group compared with the si-NC group (Figure 1(E and F)).Briefly, overexpression of KLF2 inhibited the proliferation of EC cells.

Overexpression of KLF2 inhibits invasion and migration of EC cells
The effect of KLF2 on cancer cell invasion and migration was examined using Transwell assays.The results showed that KLF2 treatment decreased invasion and migration, while si-KLF92 treatment significantly promoted invasion and migration (Figure S2(A and B)).

Overexpression of NPM1 negates the inhibitory effects of KLF2 on EC cells
Finally, to verify that KLF2 acts through NPM1 in EC, we transfected NPM1 into Ishikawa to upregulate the intracellular NPM1 mRNA level (Figure 3(A)) and co-treated with KLF2.
NPM1 level was also significantly increased (Figure 3

Discussion
Despite complete surgical resection, EC patients still face the risk of recurrence and the 5-year overall survival remains unsatisfying (Lee et al. 2017).Molecular and genomic profiling might be useful in identifying EC patients at risk of recurrence and useful to tailor appropriate adjuvant strategies in early-stage EC (Cuccu et al. 2023).Several KLFs are known to regulate cancer cell fate, including cell proliferation and survival and the tumour-initiating characteristic of cancer stemlike cells, and therefore act as either tumour suppressors or oncogenes (Zhang et al. 2015, Farrugia et al. 2016).In this study, we for the first time highlighted that KLF2 overexpression increases its binding to NPM1 promoter and therefore  suppresses NPM1 transcription, leading to inhibited EC cell growth.
KLFs have long been known to participate in uterine dysfunction in EC (Simmen et al. 2010).Existing evidence indicates KLF2 downregulation in EC (Mieszczanski 2022), which was consistent with our database prediction and in vitro assay results.Decreased KLF2 expression in primary metastatic clear cell renal cell carcinoma is in close correlation with unfavourable clinical consequences (Lu 2021).OncoLnc database revealed a similar association between KLF2 expression and the prognosis of EC patients.The upregulation of KLF2 can markedly reduce breast cancer cell viability and decrease the number of colonies formed (Yu et al. 2022).LncRNA SNHG15-mediated KLF2 knockdown facilitates pancreatic cancer cell proliferation (Zhu et al. 2019).In Ishikawa cells with relatively low KLF2 expression, KLF2 overexpression suppressed cell proliferation and reduced the number of colonies.On the contrary, KLF2 downregulation in HHEC-1-A cells with relatively high KLF2 expression accelerated cell proliferation and increased colonies formed.In terms of cancer cell migration and invasion, KLF2 upregulation weakens the migratory and invasive abilities of prostate cancer cells, while KLF2 knockdown partly annuls silencing AFAP1-AS1-exerted inhibition in gastric cancer cell viability and migration (Wang et al. 2019a, Yuan et al. 2020).Our results manifested reduced EC cell invasion and migration after KLF2 overexpression and the opposite trend after KLF2 depletion.KLF4 plays an inhibitory role in the proliferation of human EC cells (Wang et al. 2016)).KLF2 can substitute for KLF4 function in reprogramming (Parisi et al. 2010).Direct evidence about the regulation of KLF2 on EC cell growth is scarce.These findings made it plausible to suggest that KLF2 upregulation could hinder EC cell proliferation, invasion and migration in vitro.
The endoplasmic reticulum stress inducers recruit KLF16 to the cell nucleus and stimulate its interaction with NPM1 and fibrillarin in colorectal cancer (Ma et al. 2022).Similarly, our results evinced a binding relation between KLF2 and NPM1 and presented high NPM1 expression in EC cells, which paralleled a previous finding that NPM1 is upregulated in EC with the increase of clinical stages (Zhou et al. 2014).The contrary expression patterns of KLF2 and NPM1 hinted at the presence of a potential negative association, and KLF2 might participate in EC progression by regulating NPM1.As expected, KLF2 increased its enrichment in NPM1 promoter and decreased NPM1 mRNA levels.Considering that KLF2 can inhibit gene expression (Li 2021), KLF2 might bind to the NPM1 promoter and hence suppress the transcription levels of NPM1.Past research has confirmed the oncogenic role of NPM1 in estrogen-mediated EC (Shen 2022).Our results demonstrated that elevated NPM1 expression reversed the effect of KLF2 overexpression on EC cells, evidenced by potentiated Ishikawa cell proliferation, invasion, and migration.NPM1 is required for estrogen-induced endometrial proliferation, and NPM1 overexpression causes enhanced HEC-1-A and Ishikawa cell viability (Zhou 2018).miR-646 targets NPM1 mRNA to negatively regulate EC progression (Liu 2020).Collectively, NPM1 upregulation could invalidate the action of KLF2 overexpression on EC cell growth, which is the primary strength of our study.
Taken together, KLF2 shows low expression levels in EC cells, and overexpressed KLF2 decelerates EC cell growth by reducing NPM1 transcription through increased binding to NPM1 promoter.Nevertheless, there are still some limitations.The obtained results at the cellular level are limited and only one possible functional mechanism of KLF2 in EC was discussed.NPM1 mRNA levels were measured, whereas NPM1 protein levels were not included.Molecular classification and traditional clinical pathological prognostic factors are the mainstream of risk classification, which are related to personalised prognosis, clinical management, and patient treatment (Cuccu et al. 2023).Our study examined the expression of KLF2 in EC cells, whether differences in KLF2 expression exist in tissues, and whether it can become a new molecular therapy strategy that will be of great interest.Molecular/genomic analysis is the most accurate method for evaluating the prognosis of EC patients (Bogani et al. 2022).The detection of KLF2 expression in EC tissues will be included in future research to identify new and emerging categories of patients worth personalised treatment and to identify high-risk EC patients.Further studies are warranted to shed more light on the effects and functional downstream mechanisms of KLF2 in EC, the protein levels of NPM1, and the mechanisms involved, as well as result validation by in vivo experiments, to lay a theoretical foundation for novel strategies of EC treatment.
KLF2 as a transcription factor can repress gene expression(Li 2021).NPM1 expression is upregulated in EC(Zhou 2018, Liu et al. 2020).The JASPAR database predicted that KLF2 can bind to the NPM1 promoter (Figure2(A)).The Starbase database predicted a negative correlation between KLF2 and NPM1 in EC (Figure2(B)), suggesting that KLF2 may inhibit

Figure 1 .
Figure 1.Overexpression of KLF2 inhibits EC cell proliferation.A-B: the expression of KLF2 in cells was detected by RT-qPCR and Western blot; KLF2 was transfected into Ishikawa with transfected NC as the negative control, si-KLF2 was transfected into HEC-1-A with transfected si-NC as the negative control; C-D: the expression of KLF2 in cells was detected by RT-qPCR and Western blot; (E) MTT assay and (F) clonogenic assay to detect the proliferation.Three independent replicate assays were performed and data are expressed as mean ± standard deviation.t-test was used for data comparison between groups C (left), D and F; one-way ANOVA was used for data comparison between multiple groups in A-B, and C (right), and two-way ANOVA was used for data comparison between multiple groups in E. Tukey's multiple comparisons were used for all post hoc tests.
(B)).Overexpression of NPM1 resulted in increased proliferation of Ishikawa and cell colonies (Figure3(C and D)).The invasion and migration of cells in the KLF2 þ NPM1 group were also markedly increased compared to the KLF2 þ NC group (Figure3(E and F)).Overall, KLF2 inhibited NPM1 expression and thus suppressed EC cell growth.

Figure 2 .
Figure 2. KLF2 binds to the promoter region of NPM1 and inhibits NPM1 expression.A: JASPAR database to obtain the binding site of KLF2 to NPM1 promoter; B: Starbase database to predict the correlation between KLF2 and NPM1 in endometrial cancer; (C) ChIP and (D) dual luciferase assay to verify the binding of KLF2 to NPM1 promoter in EC cells; E-F: RT-qPCR to detect the NPM1 in cells mRNA expression.Three independent replicates were performed, and data are expressed as mean ± standard deviation.t-test was used for data comparison between two groups in F; one-way ANOVA was used for data comparison between multiple groups in E and two-way ANOVA was used for data comparison between multiple groups in C-D, and Tukey's multiple comparisons test was used for all post hoc tests.

Figure 3 .
Figure3.Overexpression of NPM1 negates the inhibitory effects of KLF2 on EC cells.NPM1 was transfected into Ishikawa cells, and the transfected NC was used as a negative control.The transfection efficiency of NPM1 in cells was detected by RT-qPCR (A), and then KLF2 was co-treated, and the protein expression of NPM1 in cells was detected by Western blot (A); (C) MTT method and (D) clone formation assay to detect the proliferation; E-F: Transwell assay to detect the invasion and migration.Three independent replicates were performed, and data were expressed as mean ± standard deviation.t-test was used to compare data between two groups in A, one-way ANOVA was used to compare data between multiple groups in B and D-F, and two-way ANOVA was used to compare data between multiple groups in C. Tukey's multiple comparisons test was used for all post hoc tests.