NCAPH promotes glucose metabolism reprogramming and cell stemness in ovarian cancer cells through the MEK/ERK/PD-L1 pathway

doi:10.21203/rs.3.rs-5048108/v1

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NCAPH promotes glucose metabolism reprogramming and cell stemness in ovarian cancer cells through the MEK/ERK/PD-L1 pathway

https://doi.org/10.21203/rs.3.rs-5048108/v1

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Backgrounds:

Ovarian cancer is a prevalent malignant tumors affecting the female reproductive organs with the characteristic of high heterogeneity. Non-structural maintenance of chromosomes condensin I complex subunit H (NCAPH) has been implicated in a variety of cancers.

Methods

The expression of NCAPH before or after transfection was detected using RT-qPCR and western blot. Cell stemness was assessed with spheroid formation assay. The extracellular acidification rate (ECAR) of ovarian cancer cells was appraised utilizing Seahorse Glycolysis Stress Test Assay while oxygen consumption rate (OCR) was estimated with Seahorse Mito Stress Test Assay. Lactate production and glucose consumption were evaluated with corresponding assay kits. Western blot was adopted to evaluate the contents of stem cell markers, glycolysis- and MEK/ERK/PD-L1 signaling pathway-related proteins. In vivo, the tumor size and weight were measured and KI67 expression in tumor tissues of nude mice was appraised utilizing immunohistochemical staining.

Results

It was found that NCAPH expression was upregulated in ovarian cancer cells. After silencing NCAPH expression, the stemness and glucose metabolism reprogramming were repressed. The MEK/ERK/PD-L1 signaling pathway was inhibited by NCAPH knockdown both in vitro and in vivo. NCAPH depletion was also discovered to suppress tumor growth in mice.

Conclusion

Collectively, NCAPH silence impeded the malignant progression of OC through the MEK/ERK/PD-L1 pathway.

Ovarian cancer

NCAPH

MEK/ERK/PD-L1 pathway

glucose metabolism reprogramming

stemness

Ovarian cancer (OC) has been deemed as a deadly malignancy among women, predominantly because of its high recurrence and frequent acquisition of resistance to chemotherapy in patients¹. It has been reported that over 70% OC patients were diagnosed at an advanced stage after diagnosis because the symptoms in the early stage are atypical or vague ². Currently, the treatment modalities for OC include tumor debulking surgery combined with platinum-taxane-based chemotherapy³. Despite the fact that many OC patients indeed achieve remission from standard treatment methods, cancer recurrence and chemotherapy resistance bring tremendous challenges for most patients suffering from OC^3–4. Hence, it is imperative to seek for novel and reliable therapeutic therapies for the treatment of OC.

Non-SMC condensin I complex subunit H (NCAPH) belongs to the condensin I complex, which has been recently considered as a superfamily of proteins namely kleisins⁵. NCAPH has been well-documented to be an oncogene in a variety of cancers. For example, NCAPH expression has been disclosed to be upregulated in endometrial cancer, breast cancer, and prostate cancer tissues by contrast with the normal tissues and NCAPH upregulation has close association with the poor prognosis^6–8. In addition, the silence of NCAPH can suppress stemness and glycolysis in colon adenocarcinoma⁹. Our previous study has attested that NCAPH expression is elevated in OC cells and NCAPH promotes proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in OC¹⁰. However, the role and the reaction mechanism of NCAPH in glycolysis and cell stemness in OC haven’t been investigated to date.

MEK/ERK has been implicated to play a critical role in a wide range of human cancers and participate in cell proliferation, survival, metabolism, and cell migration¹¹. Previous study demonstrated that the activation of MEK/ERK pathway can promote cell proliferation and glycolysis in breast cancer¹². Additionally, the inhibition of Raf/MEK/ERK signaling by caudatin can suppress cell stemness and glycolysis in non-small cell lung cancer cells¹³. Intriguingly, Li et al have proved that NCAPH activates MEK/ERK signaling pathway in bladder cancer to facilitate cell proliferation and suppress cell apoptosis¹⁴. Moreover, according to KEGG pathway (https://www.kegg.jp/kegg/pathway.html), MEK/ERK pathway can regulate the expression of PD-L1. Nevertheless, the mechanism of NCAPH associated with MEK/ERK/PD-L1 signaling pathway in OC remains elusive.

In summary, this study was intended to investigate the role of NCAPH in cell stemness and glycolysis in OC as well as to disclose its relationship with MEK/ERK signaling pathway, which might shed novel insights into the therapeutic strategies for OC treatment.

Cell culture and treatment

Ovarian surface epithelial cell line HOSEPiC (cat. no. MZ-7847) was provided by Ningbo Mingzhou Biotechnology Co., Ltd. and human ovarian cancer cell lines including SKOV3 (cat.no. iCell-h195), OVCAR3 (cat.no. iCell-h168), A2780 (cat.no. iCell-h004) and CAOV3 (cat.no. iCell-h037) were supplied from iCell Bioscience Inc. (Shanghai, China). All cells underwent the incubation in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin-streptomycin (both from Sigma-Aldrich). The conditions for incubation were 5% CO₂ and 37°C. To explore the mechanism of NCAPH associated with MEK/ERK/PD-L1 signaling pathway, MEK/ERK agonist LM22B-10 (50 µmol/L) was applied to treat SKOV3 cells for 1 h¹⁵ .

Cell transfection

For transfection, short hairpin RNAs (sh-RNA) specific to NCAPH (sh-NCAPH-1/2) and the corresponding scrambled sequence as a negative control (sh-NC) were constructed by GenePharma (Shanghai, China). 100 nM recombinants were introduced to SKOV3 cells by means of Lipofectamine 2000 reagent at 37 ℃ for 48 h in accordance with the recommended specifications. 48 h post-transfection, SKOV3 cells were harvested for follow-up experiments.

Reverse transcription-quantitative PCR (RT-qPCR)

The total RNA was extracted from sample SKOV3 cells with TRIzol reagent (Biosharp) and then reverse transcribed into complementary DNA (cDNA) by means of a commercial RevertAid™ cDNA Synthesis kit (Bio-Rad) in light of standard protocol. After that, the amplification of templates of was performed using SYBR Green PCR Master Mix (Takara, Toyobo, Japan) on the 7500 Fast Real-time PCR system according to the operating manual. The relative gene expression was determined with 2^−ΔΔCt method (16). The following were the sequences of primers: NCAPH forward (F), 5'-AGACGCCAAGGAAAGATGGG-3' and reverse (R), 5'-GGAACACACGCTCTGAAGGA-3' or GAPDH F, 5'-TGTGGGCATCAATGGATTTGG-3' and reverse R, 5'-ACACCATGTATTCCGGGTCAAT-3'.

Western blot

The total proteins were extracted sample SKOV3 cells with RIPA lysis buffer (Solarbio) and then bicinchoninic acid (BCA) protein assay kits (Thermo Fisher Scientific Inc.) were applied for the quantification of protein concentration in light of standard protocol. Following the separation with 8% SDS-PAGE, the proteins were shifted onto PVDF membranes, which were sealed by 5% BSA for 2 h at room temperature. Subsequently, the membranes were sequentially immunoblotted with primary antibodies targeting NCAPH (ab200659; 1:1000; Abcam), SOX2 (ab92494; 1:1000; Abcam), OCT4 (ab200834; 1:10000; Abcam), PKM2 (15822-1-AP; 1:1000; Proteintech), HK2 (ab209847; 1:1000; Abcam), LDHA (ab52488; 1:5000; Abcam), p-MEK (ab307509; 1:1000; Abcam), p-ERK (ab201015; 1:1000; Abcam), PD-L1 (28076-1-AP; 1:1000; Proteintech), MEK (11049-1-AP; 1:5000; Proteintech), ERK (ab184699; 1:10000; Abcam) or GAPDH (ab9485; 1:2500; Abcam) overnight at 4 ℃ and horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721; 1:2000; Abcam) at room temperature for 1 h. Finally, the protein bands were visualized by virtue of enhanced chemiluminescence (ECL) detection reagent (Shanghai Yeasen Biotechnology Co., Ltd. and the density of protein was assessed with ImageJ software (version 1.49, National Institutes of Health).

Spheroid formation assay

SKOV3 cells that exposed to serum-free DMEM/F12 medium introduced with B27 supplement, 20 ng/mL EGF, and 20 ng/mL bFGF (ThermoFisher Scientific, USA) in 96-well ultra-low attachment dishes were injected into 200 µL serum-free medium for at least 7 days ^17,18. With the application of a Leica DMI microscope, the number of spheres with the size bigger than 70 µm was observed.

Seahorse glycolysis stress test assay

The measurement of ECAR was estimated utilizing a Seahorse XFe96 Analyzer with Seahorse XF Glycolytic Rate Assay Kit (Agilent, cat#: 103344-100) in accordance with the recommended specifications.

Detection of ECAR and OCR

For the detection of ECAR and OCR, Seahorse XF cell Mito Stress Test Kit (Agilent, cat#: 103015-100) or Seahorse XF Glycolytic Rate Assay Kit (Agilent, cat#: 103344-100) for Agilent Seahorse XFe96 Analyzer (Agilent, California, CA, USA) were applied according to the manufacturer’s instructions. In brief, SKOV3 cells were injected into XF-96 plate and then cultivated in a Seahorse XF DMEM medium containing 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine for 1 h without CO₂ (19). Subsequently, the injection of oligomycin (final concentration 1.5 µM), luoro-carbonyl cyanide phenylhydrazone (FCCP, final concentration 1.0 µM), and rotenone/antimycin A (each final concentration 1.0 µM) into each well was implemented, following which was the evaluation of ECAR and OCR.

Detection of lactate production and glucose consumption

The production of lactate and the consumption of glucose were estimated with glucose assay kit (cat. no. CBA086; Sigma) and lactate assay kit (cat. no. K607; Biovision) according to the manufacturer’s instruction following 24 h of cultivation.

Subcutaneous tumor experiment

Prior to model construction, female nude mice (25 g, 8 weeks) that supplied from Changzhou Cavens Laboratory Animals Technology Co., Ltd. were acclimated to the experiments conditions and randomly assigned into Control, sh-NC and sh-NCAPH groups (n = 6). The right back of mice was subcutaneously injected with transfected SKOV3 cells (1×10⁶). On days 7, 10, 13, 16, 19 and 21, the size of tumor was recorded. For anesthetization, mice were forced to inhale 1–1.5% isoflurane mixed with oxygen for 4–5 min. On the 21th day, mice were euthanized by cervical dislocation for the obtaining of xenografts. The collected xenografts were weighed at once. All mice were fed freely and kept in a pathogen-free condition. The animal experiments were performed under a project license (No. G2024-160) issued by the Ethics Committee of Guangzhou Medical University. The surgical interventions and postoperative care for the animal complied with the guidelines and policies established for rodent survival surgery provided by Guangzhou Medical University.

Immunohistochemistry staining

The xenograft specimen of mice was subjected to 4% paraformaldehyde fixation, following which were dehydration, hyalinization and paraffin embedment. For immunohistochemistry staining, the sample section embedded with paraffin were exposed to dewax and rehydration. Following the blockade with BSA, the samples were probed with primary antibody targeting KI67 (cat. no. #AF0198; 1:50, Affinity) overnight at 4°C and goat anti-rabbit IgG secondary antibody (cat. no. #S0001; 1:200; Affinity) at 37°C for 1 h. With the use of an optical microscope, the immunohistochemical images were captured.

Statistical analysis

All experiments were replicated for three times. The collected experimental data were analyzed with GraphPad Prism software (version 8.0) and then presented as mean ± standard deviation. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was employed to exhibit the comparisons among multiple groups. P < 0.05 indicated statistical significance.

NCAPH expression was upregulated in ovarian cancer cells

In order to investigate the role of NCAPH in ovarian cancer, NCAPH expression was initially measured with RT-qPCR and western blot. Compared with the HOSEpiC group, the mRNA and protein expressions of NCAPH were conspicuously increased, especially in SKOV3 cells; therefore, SKOV3 cells were selected for following studies (Fig. 1A). To reduce NCAPH expression, sh-NCAPH was transfected into SKOV3 cells and the transfection efficiency was examined with RT-qPCR and western blot. Compared with the sh-NC group, the expression of NCAPH was evidently decreased, especially in sh-NCAPH-2 group; hence, sh-NCAPH-2 was employed for following studies (Fig. 1B).

NCAPH silence inhibited the stemness of ovarian cancer cells

To explore the role of NCAPH silence in the stemness of ovarian cancer cells, spheroid formation assay was initially implemented. Relative to the sh-NC group, the cell stemness was markedly reduced following the transfection of sh-NCAPH (Fig. 2A). Results obtained from western blot revealed that NCAPH interference reduced the protein expressions of SOX2 and OCT4 in SKOV3 cells by contrast with the sh-NC group (Fig. 2B).

NCAPH silence inhibited glucose metabolism reprogramming in ovarian cancer cells

Compared with the sh-NC group, NCAPH interference decreased the level of ECAR in SKOV3 cells whereas increased OCR level in comparison with the sh-NC group (Fig. 3A-B). Besides, the lactate production and glucose consumption in SKOV3 cells were inhibited following the transfection of sh-NCAPH (Fig. 3C-D). Results of the western blot demonstrated that NCAPH interference reduced the protein expressions of PKM2, HK2 and LDHA in SKOV3 cells when compared with the sh-NC group (Fig. 3E).

NCAPH silence inhibited the MEK/ERK/PD-L1 signaling pathway in ovarian cancer cells

TH effects of NCAPH silence on the expressions of proteins associated with MEK/ERK/PD-L1 signaling pathway were detected with western blot. As Fig. 4 depicted, NCAPH deficiency declined the protein expressions of p-MEK, p-ERK and PD-L1 in SKOV3 cells in contrast to the sh-NC group, indicating that NCAPH deficiency could suppress the MEK/ERK/PD-L1 signaling pathway in ovarian cancer cells.

NCAPH silence suppressed the stemness of ovarian cancer cells via the inhibition of MEK/ERK/PD-L1 signaling pathway

To explore the mechanism of NCAPH associated with MEK/ERK/PD-L1 signaling pathway, MEK/ERK agonist LM22B-10 was used to treat SKOV3 cells and above functional experiments were implemented again. Compared with the sh-NC group, NCAPH interference inhibited cell stemness, which was then partially increased by LM22B-10 treatment (Fig. 5A). In addition, the decreased protein expressions of SOX2 and OCT4 in NCAPH-silenced SKOV3 cells were increased following the treatment of LM22B-10 (Fig. 5B).

NCAPH silence suppressed glucose metabolism reprogramming in ovarian cancer cells via the inhibition of MEK/ERK/PD-L1 signaling pathway

Compared with the sh-NCAPH group, LM22B-10 treatment increased ECAR level whereas decreased OCR level in SKOV3 cells (Fig. 6A-B). Besides, the reduced lactate production and glucose consumption in NCAPH-silenced SKOV3 cells were both partially elevated by LM22B-10 treatment (Fig. 6C-D). Moreover, NCAPH interference reduced the protein expressions of PKM2, HK2 and LDHA in SKOV3 cells by contrast with the sh-NC group, while LM22B-10 treatment exhibited opposite effects on these proteins, evidenced by increased expressions of PKM2, HK2 and LDHA in LM22B-10 + sh-NCAPH group (Fig. 6E).

NAPCH silence suppressed tumor growth in mice

The appearance of mice and tumor were demonstrated in Fig. 7A. The tumor volume and weight were diminished by NCAPH interference (Fig. 7B-C). Compared with the sh-NC group, the KI67 expression was reduced following the depletion of NCAPH (Fig. 7D). Moreover, results of western blot demonstrated that silencing NCAPH reduced the protein expressions of p-MEK, p-ERK and PD-L in tumor tissues of nude mice (Fig. 7E).

At the present, the treatment methods for OC are far from satisfaction; therefore, it is profoundly vital to identify novella and reliably therapeutic targets. The present study evidenced that NCAPH silence inhibited glucose metabolism reprogramming and cell stemness in OC through the suppression of MEK/ERK/PD-L1 signaling pathway. It was also found that NCAPH silence could inhibit tumor growth of xenograft tumor mice. This preliminary study illuminated that NCAPH might be a promising therapeutic target in treating OC.

NCAPH has been acknowledged to serve as an oncogene in a variety of cancers. Take gastric cancer as an example, NCAPH expression is upregulated in gastric cancer tissue samples and cell lines (20). Besides, Zhang et al have corroborated that NCAPH expression is highly expressed in human breast cancer cell lines (21). Similarly, results of the present study showed that NCAPH expression was also conspicuously elevated in OC cell lines. Our previous study also testified that NCAPH knockdown can inhibit cell proliferation, migration and EMT in OC (10). Li et al have corroborated that NCAPH interference can suppress bladder cancer cell proliferation and significantly inhibited tumor growth in mice (14). Consistently, the tumor growth of xenograft tumor mice was also suppressed by NCAPH silence.

Cancer stem cells (CSCs), which are a subpopulation of cancer cells, have the same self-renewal and differentiation capacities as stem cells, thus promoting tumor growth and maintain the regeneration of a heterogeneous tumor mass (22, 23). Hence, CSCs are viewed to be responsible for tumor growth and advancement (24, 25). In addition, CSCs have also reported to account for chemotherapeutic resistance, thereby causing the relapse and metastasis of OC (26). Evidently, the intervention of cell stemness might be effective for the treatment of OC. A previous study illustrated that NCAPH upregulation can facilitate cell stemness in colon adenocarcinoma (9). SOX2 is pivotal for the maintainence of stem cell pluripotency and forms a complex with OCT4 (27). The overexpression of OCT4 has close relation with tumorigenicity and metastasis (28). Results of the present study revealed that the stemness of OC cells could be suppressed after interfering NCAPH expression, accompanied by decreased protein expressions of SOX2 and OCT4.

Cancer cells demonstrate a unique metabolic phenotype with the characteristic of increased glycolysis, which generates lactate (29). Glucose metabolism reprogramming is a typical hallmark of cancers (30) and the metabolic reprogramming supports the rapid proliferation of cancer cells (31). Amounting studies have attested that the modulation of glucose metabolism reprogramming can affect the progression of OC (32, 33). ECAR reflects aerobic glycolysis flux and OCR indicates the state of mitochondrial oxidative respiration (34). Results of the present study displayed that NCAPH silence decreased ECAR level whereas increased OCR level. Of note, the glycolysis in cervical cancer is suppressed by circMYC knockdown, manifested by reduced lactate production and decreased glucose consumption (35). Similarly, NCAPH depletion could diminished lactate production and glucose consumption in SKOV3 cells, indicating that NCAPH depletion could suppress glycolysis in OC.

As reported, the block of MEK/ERK signaling pathway can suppress cell stemness and glycolysis in non-small cell lung cancer or breast cancer (12, 13). Besides, according to KEGG pathway, MEK/ERK pathway can regulate PD-L1. Accumulated studies have suggested that PD-L1 is positively correlated with immune system suppression and tumor progression. For instance, the overexpression of PD-L1 trigger intracellular glycolysis through the mTOR signaling pathway, thereby promoting tumor growth and progression (36). Additionally, Lei et al have elucidated that the regulation of PD-1/PD-L1 checkpoint by circ-HSP90A accelerates cell growth, stemness, and immune evasion in non-small cell lung cancer (37). Furthermore, NCAPH has been attested to activate MEK/ERK pathway in bladder cancer to facilitate cell proliferation and inhibit cell apoptosis (14). Results of the present study showed that NCAPH silence could mediate MEK/ERK/PD-L1 signaling pathway both in vitro and in vivo and rescue experiments revealed that NCAPH deficiency inhibited cell stemness and glucose metabolism reprogramming in SKOV3 cells via MEK/ERK/PD-L1 signaling pathway.

To conclude, this study revealed the inhibitory effects of NCAPH silence on cell stemness and glucose metabolism reprogramming in SKOV3 cells and tumor growth of xenograft tumor mice. Mechanically, NCAPH functioned as a tumor promoter in OC via MEK/ERK/PD-L1 signaling pathway, mirroring that NCAPH might become a novel therapeutic target for OC.

Authors’contributions

QY and CW conceived and designed the project, QY and WA wrote the paper,

CS acquired the data, QY analyzed and interpreted the data. The authors read and approved the fnal manuscript.

Fundings

This work was supported by the Science and Technology Projects in Guangzhou(2023A03J0821), the GuangDong Basic and Applied Basic Research Foundation-Joint fund (2021A1515220055).

Availability of data and materials

All data generated or analyzed using this study was included in this published

article.

Conflict of interest

The authors declare no conflicts of interest.

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You are reading this latest preprint version

NCAPH promotes glucose metabolism reprogramming and cell stemness in ovarian cancer cells through the MEK/ERK/PD-L1 pathway

Status:

Version 1

Abstract

Backgrounds:

Methods

Results

Conclusion

Figures

Introduction

Material and methods

Cell culture and treatment

Cell transfection

Reverse transcription-quantitative PCR (RT-qPCR)

Western blot

Spheroid formation assay

Seahorse glycolysis stress test assay

Detection of ECAR and OCR

Detection of lactate production and glucose consumption

Subcutaneous tumor experiment

Immunohistochemistry staining

Statistical analysis

Results

NCAPH expression was upregulated in ovarian cancer cells

NCAPH silence inhibited the stemness of ovarian cancer cells

NCAPH silence inhibited glucose metabolism reprogramming in ovarian cancer cells

NCAPH silence inhibited the MEK/ERK/PD-L1 signaling pathway in ovarian cancer cells

NAPCH silence suppressed tumor growth in mice

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1