2022 Volume 45 Issue 12 Pages 1818-1824
Tamoxifen, which is used to treat advanced gynecological tumors, has been associated with tumor cell metastasis. Herein, we investigated the effect of tamoxifen on epithelial–mesenchymal transition in endometrial cancer and the associated signaling mechanism. Wound healing and invasion chamber assays, respectively, were performed to determine the migrative capacity and invasiveness of tamoxifen-stimulated endometrial carcinoma (RL95-2) cells. Western blotting and immunofluorescence were used to evaluate the expression of vimentin, E-cadherin, calpain 10 (CANP10), and neuropilin-1 (NRP1). Transfection of a CAPN10-harboring plasmid was used to overexpress CANP10 in RL95-2 cells, and small interfering RNAs were used to silence CANP10 and NRP1 expression. Tamoxifen induced migration, invasion, and morphological changes in RL95-2 cells. It also downregulated E-cadherin expression and upregulated vimentin, CANP10, and NRP1 expression. CANP10 silencing inhibited tamoxifen-induced NRP1 upregulation, and CANP10 or NRP1 silencing inhibited the migration and invasion of RL95-2 cells. CANP10 overexpression upregulated vimentin expression and downregulated that of E-cadherin and also increased cell migration and invasion. Silencing NRP1 protein expression inhibited the induction effect of CANP10 overexpression. In conclusion, tamoxifen promotes the epithelial–mesenchymal transition of RL95-2 cells via the CANP10/NRP1 signaling pathway. Thus, targeting CANP10 or NRP1 may be a novel strategy for preventing tamoxifen-induced endometrial cancer metastasis.
Endometrial cancer is a common gynecological tumor, posing a serious threat to women’s physical and mental health; moreover, its incidence is increasing annually. According to the WHO’s International Agency for Research on Cancer, approximately 420000 new cases of endometrial cancer were recorded in 2020 worldwide. Of these, approximately 80000 were recorded in China, accounting for approximately one-fifth of the global incidence.1,2) Endometrial cancer is mainly treated by surgery, supplemented by radiation therapy, chemotherapy, progesterone therapy, and novel targeted therapy.3–5) However, after a period of treatment, cancer cells tend to metastasize, which eventually leads to treatment failure. Therefore, suppressing the metastasis of endometrial cancer cells has important clinical significance.
Progesterone therapy is commonly used to treat endometrial cancer. Large doses of progesterone can directly act on cancer cells, inhibit the synthesis of cellular DNA and RNA, and reduce cell division, thus inhibiting cell proliferation.6,7) As a nonsteroidal anti-estrogenic drug, tamoxifen (TAM) increases the expression of progesterone receptors, and its application in combination with progesterone can improve the therapeutic effect of the latter.8,9) Recent studies have shown that TAM can increase the stemness and metastasis of estrogen receptor (ER)α36-positive breast cancer cells.10) Other studies have shown that TAM can activate G protein-coupled estrogen receptors to increase the migration and invasion of triple-negative breast cancer cells.11) These reports suggest that TAM has great potential to promote cancer. However, to date, the biological effect of TAM on endometrial cancer is not fully understood.
Epithelial–mesenchymal transition (EMT) is a biological process, wherein tumor cells undergo transformation from an epithelial to a mesenchymal phenotype.12–14) EMT enables tumor cells to acquire stronger invasive and metastatic abilities.12–14) One study has shown that TAM is associated with mutations in the EMT driver gene, TGFB2, in uterine carcinosarcoma.15) TAM can also promote EMT by upregulating c-Myc expression in endometrial cancer cells.16) It has been suggested that TAM can promote EMT in endometrial cancer. Calpain 10 (CANP10), a member of the calcium-dependent cysteine protease family, plays a positive regulatory role in the development of laryngeal cancer,17) esophageal cancer,18) and ovarian cancer.19) Clinical data show that CANP10 is also associated with poor prognosis in breast cancer.20) However, it is unclear whether CANP10 is involved in TAM-induced EMT in endometrial cancer. As a single-pass transmembrane receptor that regulates the growth, guidance, and migration of axons in the nervous system, neuropilin-1 (NRP1) plays an important role in the occurrence and development of malignant tumors.21–23) In recent years, studies have found that NRP1 plays a certain role in malignant tumor EMT.24,25) However, the relationship between NRP1 and TAM-induced EMT in endometrial cancer is still unclear, and whether it has a mediating effect with CANP10 remains unknown. Consequently, this study examined the effect of TAM on EMT in endometrial cancer cells and explored the role of CANP10/NRP1 in its pathology, as well as its underlying mechanism, to provide basic theoretical support for clinical treatment.
The human endometrial cancer cell line RL95-2 was purchased from the BeNa Culture Collection (China). The cells were placed in an incubator and maintained at 37 °C in an atmosphere of 5% CO2 to adapt to the experimental environment. On the following day, the medium was replaced with Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium (Gibco-BRL, Grand Island, NY, U.S.A.) containing 10% fetal bovine serum (FBS; Gibco-BRL) and 5 µg/mL insulin (Absin, China) for further culture.
Wound Healing AssayIn a brief, cells were scratched when they reached 100% confluence. Loose cells were washed away, and the remaining cells were treated with TAM (Sigma-Aldrich, St. Louis, MO, U.S.A.), if necessary. The scratch was photographed using a IX73 inverted microscope (Olympus, Tokyo, Japan) at 0 and 24 h.
Invasion AssayMatrigel-coated Transwell chambers were prepared for invasion assays as described previously.10)
Western BlottingCells were collected in 0.5 mL Eppendorf tubes and lysed on ice with 100 µL of radio immunoprecipitation assay (RIPA) buffer (Absin) to extract total protein. After total protein quantification, samples were prepared in a loading buffer. Subsequent experimental steps were followed as described previously.11) Primary antibodies included the following: anti-CANP10 (1 : 2000; Thermo Fisher Scientific, Waltham, MA, U.S.A.), anti-vimentin (1 : 2000; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), anti-E-cadherin (1 : 2000), anti-NRP1 (1 : 2000), anti-E-cadherin (1 : 2000), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 2000) (Cell Signaling Technology, Danvers, MA, U.S.A.). Secondary antibodies were as follows: horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary immunoglobulin G (IgG) antibody (Cell Signaling Technology). An enhanced chemiluminescence solution (Millipore, Billerica, MA, U.S.A.) was dropped onto the surface of the washed membrane, and the signals were recorded using an AI680 gel imager (GE Healthcare, Chicago, IL, U.S.A.).
Immunofluorescence StainingCells were seeded in 6-well plates to prepare the slides. On the following day, TAM treatment or direct immunofluorescence staining was performed. The cells were fixed with methanol for 15 min and blocked with 3% bovine serum albumin (BSA) for 30 min. Slides were incubated with vimentin (1 : 100) and E-cadherin (1 : 200) antibodies at 4 °C overnight. The primary antibodies were then washed off, and slides were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG and Alexa Fluor 647-conjugated goat anti-rabbit IgG secondary antibodies (Absin) for 2 h. The slides were mounted with anti-fluorescence quenching mounting solution (Absin) containing 4′,6-diamidino-2-phenylindole (0.5 µg/mL). The fluorescence was observed and recorded using a BX53 fluorescence microscope (Olympus).
Plasmid TransfectionThe pEBFP-N1 plasmid carrying CAPN10 was provided by WZ Biosciences, Inc. (China). Cells were seeded in a 12-well plate. On the following day, the plasmid was diluted with 50 µL of DMEM/F-12 medium, and 2 µL of a plasmid transfection reagent (WZ Biosciences, Inc.) was diluted with 48 µL of DMEM/F-12 medium. The mixture of the two solutions was allowed to stand for 20 min and then added to the cells, which were incubated in a fresh medium with FBS for 48 h. G418 (WZ Biosciences, Inc.) was used to select stably transfected cells.
Small Interfering RNA (siRNA) TransfectionCANP10- and NRP1-targeting siRNAs were provided by GenePharma, Inc. (China). Cells were seeded in 6-well plates. On the following day, a diluted siRNA and the corresponding negative control were mixed (1 : 1) with the diluted transfection reagent, and the mixture was allowed to stand for 20 min. The cell medium was replaced by the transfection mixture diluted with DMEM/F-12 (1 : 9). After 24–48 h of incubation, Western blotting was performed to verify protein expression for subsequent experiments.
Statistical AnalysisData were analyzed using GraphPad Prism (version 8.0) for Windows. All data are expressed as the mean ± standard deviation. Statistical significance was calculated using one-way ANOVA, followed by Fisher’s multiple comparison test, and was set at p < 0.05.
To determine the effect of TAM on the behavior of endometrial cancer cells, we observed changes in cell migration, invasion, and morphology. Different concentrations of TAM promoted the migration and invasion of RL95-2 cells (Figs. 1A, B). Cell growth changed from an aggregated to a discrete state after TAM treatment. Simultaneously, the cells changed from circular to fusiform (Fig. 1C).
A, RL95-2 cells were treated with TAM (5, 50, and 500 nM) for 24 h after scratching. Images were captured at 0 and 24 h. ** p < 0.01. B, After RL95-2 cells were treated with TAM (5, 50, and 500 nM) for 24 h, changes in cell invasion were detected using an invasion chamber assay. ** p < 0.01. C, RL95-2 cells were treated with TAM (500 nM) for 24 h, and morphological changes were observed and photographed.
We further examined the effect of TAM on vimentin and E-cadherin expression in RL95-2 cells. Western blotting showed that TAM upregulated vimentin expression and downregulated E-cadherin expression (Fig. 2A). Similar results were observed in immunofluorescence experiments (Fig. 2B).
A, RL95-2 cells were treated with TAM (5, 50, and 500 nM) for 24 h. Western blotting was used to detect the expression of vimentin and E-cadherin in cells. ** p < 0.01. B, RL95-2 cells were treated with TAM (5, 50, and 500 nM) for 24 h. Changes in vimentin and E-cadherin expression were detected by an immunofluorescence assay (×200). Scale bars: 150 µm.
Our experimental findings also showed that TAM upregulated CANP10 and NRP1 protein expression (Fig. 3A). RNA interference to suppress CANP10 expression inhibited TAM-induced upregulation of NRP1 protein expression (Fig. 3B). However, RNA interference to downregulate NRP1 expression had no effect on TAM-induced CANP10 protein expression (Fig. 3B). Interfering with CANP10 or NRP1 expression inhibited the TAM-induced downregulation of E-cadherin and upregulation of vimentin expression (Fig. 3B).
A, RL95-2 cells were treated with TAM (5, 50, and 500 nM) for 24 h. Western blotting was used to detect changes in CANP10 and NRP1 expression in cells. * p < 0.05, ** p < 0.01. B, Expression of CANP10 or NRP1 was silenced using siRNA, and RL95-2 cells were treated with TAM (500 nM) for 24 h. Western blotting was used to detect changes in the expression of CANP10, NRP1, vimentin, and E-cadherin in cells. ** p < 0.01.
To determine the roles of CANP10 and NRP1 in the TAM-induced migration and invasion of endometrial cancer cells, the inducing effect of TAM was observed after the siRNA-mediated silencing of CANP10 or NRP1. The data showed that CANP10 or NRP1 silencing inhibited the TAM-induced migration and invasion of RL95-2 cells (Figs. 4A, B).
A, RL95-2 cells with siRNA-silenced CANP10 or NRP1 expression were treated with TAM (500 nM) for 24 h after scratching. Images were captured at 0 and 24 h. ** p < 0.01. B, After RL95-2 cells with siRNA-silenced CANP10 or NRP1 expression were treated with TAM (500 nM) for 24 h, changes in cell invasion were detected using an invasion chamber assay. ** p < 0.01.
To further verify the biological role of CANP10, we overexpressed the CANP10 protein by transfecting cells with a plasmid carrying the CAPN10 gene. The data showed that CANP10 overexpression upregulated vimentin and downregulated E-cadherin protein expression (Fig. 5A) and also enhanced the migratory and invasive capacities of RL95-2 cells (Figs. 5B, C).
A, RL95-2 cells were transfected with a CANP10 plasmid and the vector, and changes in NRP1, vimentin, and E-cadherin expression were detected by Western blotting. ** p < 0.01. B, RL95-2 cells were transfected with the CANP10 plasmid and vector. Images were captured at 0 and 24 h after cell scratching. ** p < 0.01. C, RL95-2 cells were transfected with the CANP10 plasmid and vector, and changes in cell invasion were detected using an invasion chamber assay. ** p < 0.01.
Silencing NRP1 protein expression inhibited the upregulation of vimentin and downregulation of E-cadherin expression, which were induced by CANP10 overexpression (Fig. 6A), as well as the migration and invasion of RL95-2 cells (Figs. 6B, C).
A, CANP10-overexpressing RL95-2 cells were transfected with an NRP1 siRNA. Western blotting was used to determine changes in vimentin and E-cadherin expression. ** p < 0.01. B, CANP10-overexpressing RL95-2 cells were transfected with NRP1 siRNA. Images were captured at 0 and 24 h after cell scratching. p < 0.01. C, CANP10-overexpressing RL95-2 cells were transfected with NRP1 siRNA, and invasion of the cells was determined using an invasion chamber assay. ** p < 0.01.
Tumor cell EMT generally involves morphological changes, with epithelioid cancer cells losing polarity and undergoing transformation into a spindle-shaped morphology.12–14) EMT is also accompanied by increased tumor cell migration, invasion, and metastasis.12–14) In the process of EMT, the expression of cell epithelial-like markers, such as E-cadherin, cytokeratin, and ZO-1, decreases, whereas that of mesenchymal cell markers, such as N-cadherin, vimentin, and fibronectin, increases, and cells change from an epithelial to a mesenchymal phenotype.26,27) Our study demonstrated that TAM induces the transformation of endometrial cancer RL95-2 cells into a spindle-shaped morphology and increased their migratory and invasive abilities. TAM also reduced the expression of the epithelioid marker E-cadherin and increased that of the mesenchymal marker vimentin, which suggests that TAM can induce EMT in RL95-2 cells.
Furthermore, we observed that TAM treatment induced the upregulation of CANP10 protein expression. Calpains have been confirmed to be involved in the occurrence and development of various malignant tumors. Several studies have shown that calpains are involved in mediating EMT in tumor cells. Chen et al. reported that the upregulation and activation of CAPN2 induces EMT in mouse breast cancer cells.28) CAPN1 is associated with EMT in human lung epithelial cells, where it plays a regulatory role through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/transforming growth factor (TGF)-β1 signaling pathway.29) Another study showed that CAPN4 activates the Wnt/β-catenin signaling pathway, thereby inducing EMT in human melanoma cells.30) However, there has been no evidence of CANP10 involvement in EMT in endometrial cancer cells. Herein, silencing CANP10 inhibited the TAM-induced changes associated with RL95-2 cell migration and invasion, as well as the expression of EMT markers. Therefore, we postulate that TAM may mediate EMT in RL95-2 endometrial cancer cells through CANP10.
In recent years, studies have found that NRP1 plays an important role in the invasion and metastasis of malignant tumors, such as prostate, colorectal, and breast cancers.31–33) In our study, NRP1 expression was upregulated in RL95-2 cells following TAM stimulation. Moreover, silencing NRP1 inhibited TAM-induced cell migration and invasion, suggesting that NRP1 might be involved in mediating the inducing effect of TAM. A study on lung cancer showed that NRP1 can regulate EMT in lung adenocarcinoma cells and that this effect is related to the TGF-β/SMAD signaling pathway.34) In related studies on gastric cancer, NRP1 was confirmed to interact with fibronectin-1, thereby mediating the process of EMT in cells.35) Other studies have shown that NRP1 can mediate cell migration and EMT in normal endometrial cells. In our study, silencing NRP1 suppressed the TAM-induced changes in the expression of EMT markers. These results suggest that NRP1 is involved in TAM-induced EMT in RL95-2 endometrial cancer cells.
To further investigate the relationship between CANP10 and NRP1 in TAM-mediated EMT in RL95-2 cells, we silenced both and found that silencing CANP10 suppressed TAM-induced changes in NRP1 expression, whereas suppressing NRP1 expression had no effect on CANP10 expression. These data suggested that CANP10 is involved in TAM-induced EMT in RL95-2 cells through NRP1. To further confirm these findings, we overexpressed CANP10 in RL95-2 cells and found that the cells changed from an epithelial to a mesenchymal phenotype, with increased migration and invasion capacities. In addition, NRP1 silencing inhibited the induction of CANP10. These data suggest that CANP10 regulates EMT in RL95-2 endometrial cancer cells through NRP1.
Our findings support the notion that TAM can induce EMT in endometrial cancer cells and that this effect is reliant on the CANP10/NRP1 signaling pathway. Thus, the inhibition of CANP10 or NRP1 may be an effective strategy to prevent the development of TAM-induced metastasis in endometrial cancer.
Yanni Lv and Lei Xu contributed to the study design, experiments, data analysis, and manuscript writing.
The authors declare no conflict of interest.
