Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Nov 20.
Published in final edited form as: Free Radic Biol Med. 2020 Sep 11;160:775–783. doi: 10.1016/j.freeradbiomed.2020.09.010

Zinc regulates primary ovarian tumor growth and metastasis through the epithelial to mesenchymal transition

Ruitao Zhang 1,2,3,*, Guannan Zhao 2,3, Huirong Shi 1, Xinxin Zhao 4, Baojin Wang 4, Peixin Dong 5, Hidemichi Watari 5, Lawrence M Pfeffer 2,3, Junming Yue 2,3,*
PMCID: PMC7704937  NIHMSID: NIHMS1628086  PMID: 32927017

Abstract

Background:

The trace element zinc plays an indispensable role in human health and diseases including cancer due to its antioxidant properties. While zinc supplements have been used for cancer prevention, zinc is also a risk factor for cancer development. It is still unclear how zinc plays a role in ovarian cancer.

Methods:

To understand how zinc contributes to ovarian tumor growth and metastasis, we examined whether zinc contributes to tumor metastasis by regulating epithelial to mesenchymal transition (EMT) using ovarian cancer cells in vitro. Cell migration and invasion were examined using transwell plates and EMT markers were examined using Western blot. Primary ovarian tumor growth and metastasis were assessed using orthotopic ovarian cancer mouse models in vivo.

Results:

Zinc promoted EMT, while TPEN (N, N, N’, N’-tetrakis-(2-pyridylmethyl)-ethylenediamine), a membrane-permeable selective zinc chelator, inhibited EMT in a dose dependent manner in ovarian cancer cells. Moreover, zinc promoted ovarian cancer cell migration and invasion, while TPEN inhibited cell migration and invasion. Zinc activated expression of the metal response transcriptional factor-1 (MTF-1), while TPEN inhibited MTF-1 expression in a dose dependent manner. Knockout of MTF-1 inhibited zinc-induced cell migration, invasion and augmented the inhibitory effect of TPEN on cell migration and invasion. Loss of MTF-1 attenuated zinc-induced ERK1/2 and AKT activation and augmented the effect of TPEN in attenuating the ERK1/2 and AKT pathways. TPEN effectively inhibited primary ovarian tumor growth and metastasis in an orthotopic ovarian cancer mouse model by suppressing EMT.

Conclusion:

zinc contributes to ovarian tumor metastasis by promoting EMT through a MTF-1 dependent pathway. Zinc depletion by TPEN may be a novel approach for ovarian cancer therapy by inhibiting EMT and attenuating the ERK1/2 and AKT pathways.

Keywords: Zinc, TPEN, MTF-1, EMT, ovarian carcinoma, metastasis, invasion

Graphical Abstract

Graphical Abstract

graphic file with name nihms-1628086-f0001.jpg

1. Introduction

Ovarian cancer is one of the most lethal gynecological malignancies due to pelvic and abdominal metastasis. Although a combination of approaches are used for ovarian cancer therapy, including debulking surgery, chemotherapy and targeted therapy, the overall survival rate is still low [1, 2]. Metastatic ovarian tumors account for 75% ovarian malignancies, and tumor cells detach from the ovaries and spread into peritoneal organs including liver, spleen, and intestine. The molecular mechanisms underlying ovarian tumor metastasis remain unclear. However, accumulating evidence indicates that the epithelial to mesenchymal transition (EMT) is associated with ovarian tumor metastasis. During ovarian tumor metastasis, epithelial tumor cells enter into the peritoneal cavity and form spheroids with a mesenchymal phenotype, then disseminated into distant organs with a phenotypic switch to epithelial cells, which promotes cell proliferation, migration, and metastasis to distant organs [3]. Targeting EMT for cancer therapy will reverse the EMT process, and thus inhibit tumor metastasis. There are multiple clinical drugs for cancer therapy by inhibiting EMT [4, 5].

Zinc is a trace element that plays a vital role in cell growth, differentiation and apoptosis. Zinc is required by more than 300 enzymes and nearly 2000 transcription factors, and is associated with multiple human diseases due to its role in neutralize the free radicals and antioxidant properties [6], including different types of cancer [7-16]. The ratio of copper vs zinc and CA125 (Cancer Antigen 125) are biomarkers for several different cancers including ovarian cancer [17-21]. Zinc supplements have been used for cancer prevention [22-24]. Zinc has been shown to inhibit the proliferation of esophageal squamous cell carcinoma cells [25]. However, dietary zinc supplement is also a potential risk factor by activating the zinc transporter in breast cancer [26] and prostate cancer [27]. Zinc promotes EMT in colorectal and lung cancers [28, 29], suggesting its oncogenic role in both cancer types. All those studies indicated that zinc plays different roles depending on cancer types. The role of zinc in ovarian cancer has not been well investigated.

Zinc activates the metal response element-binding transcription factor-1 (MTF-1), a cellular zinc sensor, which maintains zinc homeostasis by directly binding to the highly conserved six-zinc-figurer domains [30]. MTF1 protects oxidative stress by transcriptionally regulates downstream target genes through metal response elements (MREs) such as metallothionein (MT) [31, 32]. We previously reported that MTF-1 functioned as an oncogene in ovarian cancer by promoting EMT[33]. Therefore, we hypothesize that zinc may promote EMT by activating MTF-1 in ovarian cancer cells, and hence depletion of zinc might provide a novel therapeutic approach for ovarian cancer.

TPEN (N, N, N', N'-tetrakis-(2-pyridylmethyl)-ethylene-diamine) is a membrane-permeable zinc selective chelator, which has been used to deplete intracellular zinc in cancer cells [34, 35]. Several studies indicated that TPEN induced cell apoptosis and inhibited cell proliferation in variety of cancer types [36-40]. Variants of TPEN have been synthesized to improve stability, efficacy and reduce toxicity, and showed efficacy in breast cancer [41]. TPEN may inhibit tumor metastasis by targeting EMT through zinc chelation.

In this study, we determined the role of zinc in ovarian cancer by examining the EMT phenotypic switch following ZnSO4 and TPEN treatment, and further examined whether zinc activates MTF-1 in ovarian cancer cells. In addition, we tested the therapeutic potential of TPEN in orthotopic ovarian cancer mouse models.

2. Materials and Methods

Cell culture

The ovarian cancer cell lines OVCAR-3 and OVCAR-8 (obtained from National Cancer Institute) were cultured in RPMI 1640 medium supplemented with 10% FBS (Hyclone, Logan, UT), 100 U/ml penicillin, and 100μg/ml streptomycin (Invitrogen, Carlsbad, CA). TPEN and ZnSO4 were purchased from Sigma (St. Louis, MO). TPEN was dissolved in absolute alcohol and diluted in DPBS (Mediatech, Manassas, VA), and ZnSO4 was diluted in DPBS.

Cell migration assays

Transwell migration assays were performed using modified chambers (BD Falcon, San Jose, CA) as described previously [42]. Briefly, OVCAR-3 and OVCAR-8 cells were pretreated with or without TPEN or ZnSO4 for 24h. Cells (3×105) in 300μl serum free medium with or without TPEN or ZnSO4 were added to the upper chamber and 10% FBS in medium as chemoattractant in the bottom chamber. Cells were incubated for 12h. Migrated cells were stained with Crystal Violet and counted from at least three different fields using Image J software.

Cell invasion assays

Cell invasion assays were performed as described previously [42]. Briefly, OVCAR-3 and OVCAR-8 cells were pretreated with or without TPEN or ZnSO4 for 24h. Cells (3×105) were seeded in serum-free medium with or without TPEN and ZnSO4 into Matrigel (BD BioCoat) precoated inserts in 24-well plates. The invaded cells were stained with Hematoxylin and Eosin (H&E) and counted from at least three different fields.

Zinc staining

To assess zinc uptake, we stained MTF-1 knockout (KO) and control ovarian cancer cells following zinc and TPEN treatment. Briefly, cells were seeded in 24-well plates at a density of 5×103 viable cells per well and treated with 20μM ZnSO4 for 30min or 0.6μM TPEN for 6h in serum free medium at 37°C. Cells were incubated with 1 μM FluoZin-3-AM (Fisher Scientific) in a buffer (25mM HEPES [pH 7.35], 120 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1.3 mM CaCl2, 1 mM MgCl2, 1 mM NaH2PO4, 0.3% BSA) for 30 min at 37°C, and then cells were washed twice using this buffer without BSA, and imaged using a digital Nikon fluorescent microscope at 40× magnification.

Western blot

Ovarian cancer cells were collected in RIPA buffer (Thermo Scientific; Rockford, IL) containing 1% halt proteinase inhibitor cocktail (Thermo Scientific). An equal amount of protein (40μg/lane) was loaded onto 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk for 1h, and incubated with primary antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1000, Sigma, St. Louis, MO), MTF-1(1:500, Novus Biologicals), E-cadherin, cytokeratin-7, beta-catenin, vimentin, total-ERK1/2, phospho-ERK1/2, total-AKT or phospho-AKT (1:1000, Cell Signaling). Band intensity was measured using Image J software.

Mouse xenograft model

Animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center. OVCAR-8 cells (1×106 ) transduced with a lentiviral vector expressing luciferase (pLenti-UBC-Luc2) were intrabursally injected into two-month-old immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) female mice. Five mice were used in each experimental group. Tumor initiation and progression were monitored using a Xenogen animal imaging system once a week. At one week following cell injection, mice were intraperitoneally injected with TPEN (diluted with Ethanol: Glycerol: Water=1:3:6) at 1mg/Kg body weight every other day. All mice were sacrificed at the fourth week following cell injection, the tumors were weighed and processed for Western blot, H&E staining or immunostaining.

Immunofluorescence staining

Immunofluorescent staining was performed as described previously [43]. To detect the expression of MTF-1 and EMT markers in ovarian tumor tissues, tumor sections from three different mice were incubated with different primary antibodies against MTF-1 (Rockland, 1:200), cytokeratin-7, vimentin (Cell signaling, 1:200), and PCNA (1:200, Santa Cruz) were incubated at 4°C overnight. Alexa 594 or 488 conjugated goat anti-rabbit or mouse antibodies were incubated (1:200 dilution; ThermoFisher Scientific, Inc.) for 1h at room temperature. Nuclei were counterstained with DAPI (Vector Laboratories). Images were taken using a Nikon Ti inverted fluorescence microscope (Nikon Corporation).

Statistical analysis

Significant differences were determined from at least two independent experiments performed in triplicate by one-way analysis of variance (one-way ANOVA) and Student’s t-test, and data were presented as mean ± SD. In all tests, the P-values less than 0.05 was considered as statistically significant.

3. Results

Zinc promotes EMT, migration and invasion in ovarian cancer cells

We previously showed that knockout of MTF-1 resulted in the inhibition of EMT in ovarian cancer cells[33]. To examine whether zinc promotes EMT by activating MTF-1, we treated ovarian cancer cell OVCAR-3 and OVCAR-8 for 48h with different doses of ZnSO4. MTF-1 expression was induced by zinc in a dose dependent manner. In addition, epithelial cell markers E-cadherin and cytokeratin-7 were downregulated following zinc treatment, and mesenchymal markers beta-catenin and vimentin were upregulated in a dose-dependent manner as shown by Western blot (Figure 1A, B), indicating that zinc promoted EMT in ovarian cancer cells and fibroblast-like cell morphologies were observed following zinc treatment (Figure S1A, B).

Figure 1. Zinc promoted EMT, migration and invasion in ovarian cancer cells.

Figure 1.

Open in a new tab

A,B. Western Blot analysis of MTF-1 and EMT marker expression in OVCAR-3 (A) and OVCAR-8 cells (B) following different doses of ZnSO4 treatment for 48h. C. Transwell cell migration assay of OVCAR-3 and OVCAR-8 cells treated for 24h using 20μM ZnSO4 and migrated cells were stained with crystal violet. D. Matrigel coated transwell invasion assay of OVCAR-3 and OVCAR-8 cells treated with 20μM ZnSO4 for 24 h and H&E stained. (***p<0.001, *p<0.05).

Since zinc induced EMT, we determined the effect of zinc on cell migration and invasion in OVCAR-3 and OVCAR-8 cells. Cells were treated with ZnSO4 for 24h, and cell migration was examined using transwell plates and invasion was assessed using Matrigel coated transwell plates. Cell migration and invasion in both OVCAR-3 and OVCAR-8 cells were significantly increased by zinc treatment compared to the vehicle-treated cells (Figure 1C, D). Our data indicated that zinc contributed to ovarian tumor invasiveness by promoting EMT in ovarian cancer cells.

TPEN inhibits EMT, migration and invasion in ovarian cancer cells

To further examine the role of zinc on EMT, we treated ovarian cancer cells (OVCAR-3 and OVCAR-8) with different doses of the selective zinc chelator TPEN for 48h, and then MTF-1 and EMT markers were determined by Western blot. In contrast to the effects of zinc addition, MTF-1 expression was reduced in a dose dependent manner following TPEN treatment. In addition, the epithelial markers E-cadherin and cytokeratin-7 were significantly upregulated by TPEN treatment, while the mesenchymal markers beta-catenin and vimentin were downregulated (Figure 2A, B), indicating that TPEN inhibited EMT in ovarian cancer cells (Figure 2A, B). We also assessed the efficacy of TPEN in inhibiting cell migration and invasion. Ovarian cancer OVCAR-3 and OVCAR-8 cells were treated with TPEN for 24h, then cell migration and invasion were examined using transwell assays. TPEN significantly reduced cell migration and invasion in both OVCAR-3 and OVCAR-8 cells compared to the vehicle treated cells (Figure 2C, D).

Figure 2. TPEN inhibited EMT, migration and invasion in ovarian cancer cells.

Figure 2.

Open in a new tab

A,B. Western Blot analysis of MTF-1 and the indicated EMT markers in OVCAR-3 (A) and OVCAR-8 cells (B) following different doses of TPEN treatment for 48 h. C. Transwell cell migration assay of OVCAR-3 and OVCAR-8 cells treated with 0.6μM TPEN for 24h, and the migrated cells were stained with crystal violet. D. Matrigel coated transwell invasion assay of OVCAR-3 and OVCAR-8 cells treated with 0.6μM TPEN for 24h and H&E stained. (***p<0.001, **p<0.01).

Loss of MTF-1 reduced zinc induced ovarian cancer cell migration and invasion

Zinc promotes ovarian cancer cell migration and invasion by activating MTF-1 expression. To further determine whether MTF-1 plays a role on the function of zinc in ovarian cancer cells, we generated MTF-1 knockout and control OVCAR-3 and OVCAR-8 cells using lentiviral CRISPR/Cas9 nickase vector as we reported previously [33]. MTF-1 KO and control OVCAR-3 and OVCAR-8 cells were treated with ZnSO4 for 24h and intracellular zinc was examined using zinc labeled fluorescent dye FluoZin-3-AM. Knockout of MTF-1 significantly reduced intracellular zinc accumulation in both cell lines as shown by zinc fluorescent staining (Figure S2A, B) and loss of MTF-1 expression was validated by Western blot (Figure S3A, B). Cell migration and invasion were also examined using transwell plates. Knockout of MTF-1 significantly inhibited zinc induced cell migration (Figure 3A, B) and invasion (Figure 3C, D) in both OVCAR-3 and OVCAR-8 cells. Taken together, our data demonstrated that knockout of MTF-1 antagonized zinc-induced ovarian tumor cell migration and invasion.

Figure 3. Knockout of MTF-1 inhibited zinc-induced cell migration and invasion.

Figure 3.

Open in a new tab

A, B. Transwell cell migration assay of MTF-1 KO and control OVCAR-3 (A) and OVCAR-8 cells (B) following 20μM ZnSO4 treatment for 24h. C, D. Matrigel coated transwell invasion assays of MTF-1 KO and control OVCAR-3(C) and OVCAR-8 cells (D) following 20μM ZnSO4 treatment for 24h. (***p<0.001, **p<0.01).

Loss of MTF-1 augments the inhibition of ovarian cancer cell migration and invasion mediated by TPEN

TPEN attenuates zinc signaling through chelation and reducing the intracellular zinc concentration. We also examined intracellular zinc levels by FluoZin-3-AM staining following TPEN treatment in MTF-1 KO and control cells. Loss of MTF-1 further augmented zinc depletion induced by TPEN as shown by fluorescent staining (Figure S4A, B) and MTF-1 expression was further reduced by TPEN (Figure S5A, B). We also assessed cell migration and invasion by treating MTF-1 KO and control OVCAR-3 and OVCAR-8 cells with TPEN for 24h. Loss of MTF-1 significantly augmented the inhibition of cell migration (Figure 4A, B) and invasion induced by TPEN (Figure 4C, D) as compared to control cells.

Figure 4. Loss of MTF-1 augmented the inhibitory effect of TPEN on cell migration and invasion.

Figure 4.

Open in a new tab

A, B. Transwell cell migration assay of MTF-1 KO and control OVCAR-3 (A) and OVCAR-8 cells (B) following 0.6μM TPEN treatment for 24h. C, D. Matrigel coated transwell invasion assays of MTF-1 KO and control OVCAR-3(C) and OVCAR-8 cells (D) following 0.6μM TPEN treatment for 24h (***p<0.001, **p<0.01).

Zinc activates cellular survival pathways ERK1/2 and AKT and TPEN attenuates zinc activated both pathways in ovarian cancer cells

In ovarian cancer cells, we found that zinc activated MTF-1 and promoted EMT while TPEN inhibited MTF-1 and EMT. To elucidate the molecular mechanisms underlying zinc-mediated EMT in ovarian cancer cells, we examined the effect of zinc on two cellular survival pathways, ERK1/2 and AKT in OVCAR-3 and OVCAR-8 cells. Zinc activated the EKR1/2 and AKT pathways in both cell lines following different doses of ZnSO4 treatment (Figure 5A and B). In contrast, TPEN attenuated both pathways in a dose dependent manner (Figure 5C and D) in both cell lines. Next, we examined whether TPEN blocked zinc induced EKR1/2 and AKT activation by pretreating OVCAR-3 and OVCAR-8 cells with TPEN, followed by treatment with ZnSO4. TPEN significantly attenuated the activation of the EKR1/2 and AKT pathways by zinc in both cell lines (Figure 5E and F).

Figure 5. Zinc activated ERK1/2 and AKT signaling, while TPEN attenuated both pathways.

Figure 5.

Open in a new tab

A. B. Western Blot analysis of phosphorylated and total EKR1/2 and AKT in OVCAR-3 (A) and OVCAR-8 (B) following different doses of ZnSO4 treatment for 20min. C, D. Western Blot analysis of phosphorylated and total EKR1/2 and AKT in OVCAR-3 (C) and OVCAR-8 (D) following different doses of TPEN treatment for 6 h and 24h, respectively. E, F, Western blot analysis of phosphorylated and total EKR1/2 and AKT in OVCAR-3 (E) and OVCAR-8(F) cells following 0.6μM TPEN treatment for 6h and 24h, respectively and then treated with 20μM ZnSO4 for 20min (***p<0.001).

Loss of MTF-1 attenuated zinc induced ERK1/2 and AKT activation in ovarian cancer cells

We previously reported that loss of MTF-1 attenuates the EKR1/2 and AKT pathways in ovarian cancer cells [33]. To examine whether MTF-1 is involved in zinc-induced EMT through ERK1/2 and AKT pathways, we treated MTF-1 KO and control OVCAR-3 and OVCAR-8 cells with ZnSO4 and examined activation of the ERK1/2 and AKT pathways by Western blot. Knockout of MTF-1 attenuated zinc induced ERK1/2 and AKT pathways as determined by their phosphorylation in both OVCAR-3 and OVCAR-8 cells (Figure 6A and B). We further examined the effect of TPEN treatment on both pathways in MTF-1 KO and control OVCAR-3 and OVCAR-8 cells. We found that loss of MTF-1 augmented the TPEN induced attenuation of both EKR1/2 and AKT pathways (Figure 6C and D). Our data indicated that the zinc/MTF-1 axis contributes to the activation of the ERK1/2 and AKT pathways while TPEN inhibits both pathways in ovarian cancer cells.

Figure 6. Loss of MTF-1 attenuated zinc induced EKR1/2 and AKT activation in ovarian cancer cells.

Figure 6.

Open in a new tab

A, B. Western Blot analysis of phosphorylated and total EKR1/2 and AKT in MTF-1 KO and control OVCAR-3 (A) and OVCAR-8 (B) following 20μM ZnSO4 treatment for 20 mins. C, D. Western Blot analysis of phosphorylated and total EKR1/2 and AKT in MTF-1 KO and control OVCAR-3 (C) and OVCAR-8 (D) following 0.6 μM TPEN treatment for 6h.and 24h, respectively (***p<0.001).

TPEN inhibits primary ovarian tumor growth and metastasis in orthotopic ovarian cancer mouse models

TPEN inhibits EMT, cell migration and cell invasion in ovarian cancer cells, indicating that TPEN may suppress ovarian tumor metastasis. To examine the efficacy of TPEN in ovarian cancer therapy, we intrabursally injected luciferase labelled ovarian cancer OVCAR-8 cells into immunocompromised NSG female mice and treated the mice with TPEN after tumor initiation. Xenogen live animal imaging was used to monitor tumor growth and metastasis. Primary ovarian tumor growth and metastasis was significantly reduced in TPEN-treated mice as compared to vehicle-treated mice at one month following TPEN treatment (Figure 7A). In addition, ovarian tumors were significantly smaller in TPEN-treated mice as compared to vehicle-treated mice (Figure 7B). TPEN inhibited MTF-1 and the mesenchymal markers beta-catenin and vimentin, while TPEN upregulated the epithelial markers E-cadherin and cytokeratin-7 in primary ovarian tumors as shown by Western blot and immunostaining (Figure 7C, Figure S6). In addition, we found that the EKR1//2 and AKT pathways were significantly attenuated in primary ovarian tumors of TPEN treated mice (Figure 7D). TPEN treatment not only inhibited primary ovarian tumor, but also inhibited ovarian tumor metastasis. Metastatic tumors were observed in distant organs such as the liver, spleen and kidney of vehicle-treated mice, but were rarely found in those organs of TPEN-treated mice as shown by bioluminescent imaging (Figure 8A). Metastatic tumors in these organs were also confirmed by H&E staining (Figure 8B). Taken together, our data indicated that TPEN inhibited primary ovarian tumor growth and metastasis by suppressing EMT through attenuating the EKR1/2 and AKT pathways.

Figure 7.

Figure 7.

Open in a new tab

A. Primary ovarian tumor and tumor dissemination in mice was monitored with Xenogen live animal imaging following intrabursal injection of OVCAR8 cells and treated for five days per week with TPEN (1 mg/Kg body weight) or vehicle (DMSO) (n=5). Bioluminescent image was taken at one month following TPEN treatment. B. Primary ovarian tumors were collected at necropsy after 1 month of treatment and weighed. C. Western blot analysis of the MTF-1 and indicated EMT markers in ovarian tumors of three different mice. D. Western blot analysis of phosphorylated and total EKR1/2 and AKT in ovarian tumors from three different mice treated with TPEN or vehicle. (***p<0.001, **p<0.01).

Figure 8. TPEN inhibited ovarian tumor metastasis in orthotopic ovarian cancer mouse models.

Figure 8.

Open in a new tab

A. Metastatic tumors in liver, spleen and kidney were identified by bioluminescence in five different mice treated with TPEN or vehicle. B. Metastatic tumors in liver, spleen and kidney were H&E stained. (**p<0.01, *p<0.05).

4. Discussion

Although zinc supplementation has been used for cancer prevention, excessive amounts of zinc is potentially a risk factor to induce carcinogenesis. In this study, we demonstrated for the first time that zinc promoted EMT in ovarian cancer cells by inducing MTF-1 expression and activating the downstream ERK1/2 and AKT pathways, suggesting that zinc may facilitate ovarian tumor metastasis. We also found that depletion of zinc in ovarian cancer cells with TPEN inhibited EMT by downregulating MTF-1 expression and attenuating both then EKR1/2 and AKT pathways. Furthermore, TPEN effectively inhibited primary ovarian tumor growth and metastasis, indicating that zinc depletion has therapeutic potential for ovarian cancer therapy by suppressing EMT.

The role of zinc in cancer is somewhat controversial. Zinc supplementation inhibited prostate cancer growth at low levels (80 mg/day) and promoted prostate cancer at high levels (100 mg/day) [44], but had no obvious effect in head and neck cancer [45, 46]. Zinc was shown to inhibit esophageal squamous cell carcinoma proliferation, which was reversible by TPEN [25]. Zinc supplementation in combination with chemotherapy and radiotherapy attenuated local tumor recurrence and improved the overall survival of patients with advanced nasopharyngeal cancer [47]. Zinc has reported anticancer efficacy in non-small-cell lung cancer cells in both p53-wild-type and p53-deficient cancer cells [48]. However, zinc promoted EMT in colorectal cancer cells and lung cancer cells [28, 29], which was consistent with our findings that zinc promoted EMT and TPEN reversed zinc-induced EMT in ovarian cancer cells. Taken together, these studies indicate that zinc can inhibit or promote tumor cell growth in different cancer types. Although the EMT phenotypic switch is regulated by multiple transcriptional factors in cancer cells, our study demonstrated that zinc also regulated EMT in ovarian cancer cells. Thus, controlling intracellular zinc concentration apparently plays an important role in ovarian tumor initiation, progression and metastasis.

MTF-1 is a zinc sensor, which is activated by zinc through its binding to the six highly conserved zinc finger domains[49]. There are MRE sequences in the promoter of MTF1 responsive to zinc treatment[50]. We established stable MTF-1 KO ovarian cell lines with lentiviral CRISPR/Cas9nickase vector[33]. Loss of MTF-1 significantly reduced intracellular zinc levels, indicating that zinc concentrations are positively correlated with MTF-1 expression in ovarian cancer cells. Our finding is consistent with a previous study showing that zinc induces MTF1, Znt1 and MT expression in IPEC-J2 and Caco-2 cells and MTF1 expression level corresponds with intracellular zinc concentration although different cell types respond differently to zinc treatment[51]. We further showed that loss of MTF-1 augmented the TPEN-induced zinc depletion. Interestingly, a previous study showed that zinc has antitumor activity in melanoma by inhibiting the expression of the HIF1α transcriptional factor, but not MTF-1[52], suggesting that MTF-1 is not responsive to zinc in melanoma. Zinc induced MTF1 expression in p53 expressing cells, but not in the p53 inactivated breast cancer cells[53]. Ovarian cancer OVCAR3 and OVCAR8 cells are expressing mutant p53 and MTF1expression was induced by zinc treatment although we didn’t test whether zinc induced MTF1 expression in p53 null ovarian cancer cells. However, we showed that zinc promoted EMT by activating MTF-1 expression in ovarian cancer cells, indicating that zinc promotes ovarian cancer tumorigenesis. MTF-1 regulates more than 1000 genes by binding to MREs in response to zinc [32]. Our study indicates a new strategy for ovarian cancer treatment by depleting zinc using TPEN. Indeed, TPEN inhibited ovarian tumor growth and metastasis with a dose of 10 mg/Kg body weight. Previous studies showed that mice were well tolerated when TPEN was administrated to mice with 10mg/Kg body weight intraperitoneally while it was toxic if mice were dosed over 30mg/Kg [54]. Therefore, TPEN didn’t cause any toxicity in our mouse models and no significant body weight loss was observed.

The cell signaling pathway by which zinc induces EMT in different types of cancers is relatively unknown. GSK/mTOR pathways were found to contribute to zinc-induced EMT in colorectal cancer [28]. FAK, Rac1 and RhoA were involved in zinc-mediated EMT in lung cancer[29]. We previously showed that MTF-1 regulated EMT in ovarian cancer cells by activating both the ERK1/2 and AKT pathways [33]. Herein we demonstrated that zinc activated the ERK1/2 and AKT pathways by inducing MTF-1 expression. In contrast, TPEN treatment of ovarian cancer cells inhibited MTF-1 expression and attenuated both the ERK1/2 and AKT pathways, and loss of MTF-1 attenuated zinc-induced ERK1/2 and AKT activation. Our data indicated that the zinc/MTF-1 axis contributed to EMT in ovarian cancer cells through activation of the EKR1/2 and AKT pathways. Previous studies indicated that zinc induced MTF1 expression and activated ERK1/2 in mouse embryo fibroblasts How MTF-1 directly contributes to zinc-induced ERK1/2 and AKT activation, which requires further investigation in ovarian cancer. In orthotopic ovarian cancer mouse models, we also found that tumors from TPEN treated mice showed inhibition of EMT and the attenuation of the EKR1/2 and AKT pathways, while MTF-1 expression was also decreased in tumors from TPEN-treated mice.

5. Conclusions

We presented evidence that zinc was a potential risk factor and promoted EMT in ovarian cancer cells by activating MTF-1 and the downstream ERK1/2 and AKT pathways. TPEN has the therapeutic potential for ovarian cancer therapy by reversing EMT through zinc mediated cellular survival signaling.

Supplementary Material

1

  • Zinc promotes ovarian cancer cell migration, invasion and epithelial to mesenchymal transition.

  • Zinc activates MTF1 expression and promotes ERK1/2 and AKT activation.

  • Zinc chelator TPEN reverse the zinc induced EMT, inhibits MTF1 expression and attenuates ERK1/2 and AKT pathways.

  • TPEN inhibits ovarian tumor growth and metastasis in orthotopic ovarian cancer mouse models.

Acknowledgments

Funding: This work was supported by a grant (1R21CA216585-01A1) from National Institute of Health to Y.J. and in part by the Health and Family Planning Technology Overseas Training Project (2018028) of Henan Province, China and by Scientific and technological projects (202102310392) of Henan Province, China to Z.R.

Abbreviations:

EMT:

epithelial to mesenchymal transition

MTF-1:

metal response element-binding transcription factor-1

KO:

knockout

MRE:

metal response elements

MT:

metallothionein (MT)

TPEN:

N, N, N’, N’-tetrakis-(2-pyridylmethyl)-ethylenediamine

NSG:

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ

H.E:

Hematoxylin and Eosin

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Availability of data and materials: Not applicable.

Ethics approval and consent to participate:

Not applicable.

Consent for publication

Not applicable.

Conflicts of Interest: The authors declare no conflict of interest.

References:

  • 1.Siegel RL, Miller KD, Jemal A: Cancer statistics, 2019. CA Cancer J Clin 2019, 69(1):7–34. [DOI] [PubMed] [Google Scholar]
  • 2.Miller KD, Nogueira L, Mariotto AB, Rowland JH, Yabroff KR, Alfano CM, Jemal A, Kramer JL, Siegel RL: Cancer treatment and survivorship statistics, 2019. CA Cancer J Clin 2019. [DOI] [PubMed] [Google Scholar]
  • 3.Davidson B, Trope CG, Reich R: Epithelial-mesenchymal transition in ovarian carcinoma. Front Oncol 2012, 2:33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Davis FM, Stewart TA, Thompson EW, Monteith GR: Targeting EMT in cancer: opportunities for pharmacological intervention. Trends Pharmacol Sci 2014, 35(9):479–488. [DOI] [PubMed] [Google Scholar]
  • 5.Deng J, Wang L, Chen H, Hao J, Ni J, Chang L, Duan W, Graham P, Li Y: Targeting epithelial-mesenchymal transition and cancer stem cells for chemoresistant ovarian cancer. Oncotarget 2016, 7(34):55771–55788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Singh CK, Pitschmann A, Ahmad N: Resveratrol-zinc combination for prostate cancer management. Cell Cycle 2014, 13(12): 1867–1874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Prasad AS: Discovery of human zinc deficiency: 50 years later. J Trace Elem Med Biol 2012, 26(2-3):66–69. [DOI] [PubMed] [Google Scholar]
  • 8.Schwingshackl L, Boeing H, Stelmach-Mardas M, Gottschald M, Dietrich S, Hoffmann G, Chaimani A: Dietary Supplements and Risk of Cause-Specific Death, Cardiovascular Disease, and Cancer: A Systematic Review and Meta-Analysis of Primary Prevention Trials. Adv Nutr 2017, 8(1):27–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Mohammadifard N, Humphries KH, Gotay C, Mena-Sanchez G, Salas-Salvado J, Esmaillzadeh A, Ignaszewski A, Sarrafzadegan N: Trace minerals intake: Risks and benefits for cardiovascular health. Crit Rev Food Sci Nutr 2017:1–13. [DOI] [PubMed] [Google Scholar]
  • 10.Li L, Gai X: The association between dietary zinc intake and risk of pancreatic cancer: a meta-analysis. Biosci Rep 2017, 37(3). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Joshaghani H, Mirkarimi HS, Besharat S, Roshandel G, Sanaei O, Nejabat M: comparison of the Serum Levels of Trace Elements in Areas with High or Low Rate of Esophageal Cancer. Middle East J Dig Dis 2017, 9(2):81–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Singh BP, Dwivedi S, Dhakad U, Murthy RC, Choubey VK, Goel A, Sankhwar SN: Status and Interrelationship of Zinc, Copper, Iron, Calcium and Selenium in Prostate Cancer. Indian J Clin Biochem 2016, 31(1):50–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Pramchu-em C, Meksawan K, Chanvorachote P: Zinc Sensitizes Lung Cancer Cells to Anoikis through Down-Regulation of Akt and Caveolin-1. Nutr Cancer 2016, 68(2):312–319. [DOI] [PubMed] [Google Scholar]
  • 14.Schwingshackl L, Hoffmann G, Buijsse B, Mittag T, Stelmach-Mardas M, Boeing H, Gottschald M, Dietrich S, Arregui M, Dias S: Dietary supplements and risk of cause-specific death, cardiovascular disease, and cancer: a protocol for a systematic review and network meta-analysis of primary prevention trials. Syst Rev 2015, 4:34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Prasad AS: Impact of the discovery of human zinc deficiency on health. J Trace Elem Med Biol 2014, 28(4):357–363. [DOI] [PubMed] [Google Scholar]
  • 16.Kolenko V, Teper E, Kutikov A, Uzzo R: Zinc and zinc transporters in prostate carcinogenesis. Nat Rev Urol 2013, 10(4):219–226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Gal D, Lischinsky S, Friedman M, Zinder O: Prediction of the presence of ovarian cancer at surgery by an immunochemical panel: CA 125 and copper-to-zinc ratio. Gynecol Oncol 1989, 35(2):246–250. [DOI] [PubMed] [Google Scholar]
  • 18.Gao ZJ: [Diagnostic value of serum copper/zinc ratio in gynecologic tumors]. Zhonghua Zhong Liu Za Zhi 1988, 10(6):434–436. [PubMed] [Google Scholar]
  • 19.Stepien M, Jenab M, Freisling H, Becker NP, Czuban M, Tjonneland A, Olsen A, Overvad K, Boutron-Ruault MC, Mancini FR et al. : Pre-diagnostic copper and zinc biomarkers and colorectal cancer risk in the European Prospective Investigation into Cancer and Nutrition cohort. Carcinogenesis 2017, 38(7):699–707. [DOI] [PubMed] [Google Scholar]
  • 20.Stepien M, Hughes DJ, Hybsier S, Bamia C, Tjonneland A, Overvad K, Affret A, His M, Boutron-Ruault MC, Katzke V et al. : Circulating copper and zinc levels and risk of hepatobiliary cancers in Europeans. Br J Cancer 2017, 116(5):688–696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Vella V, Malaguarnera R, Lappano R, Maggiolini M, Belfiore A: Recent views of heavy metals as possible risk factors and potential preventive and therapeutic agents in prostate cancer. Mol Cell Endocrinol 2017, 457:57–72. [DOI] [PubMed] [Google Scholar]
  • 22.Mansouri A, Hadjibabaie M, Iravani M, Shamshiri AR, Hayatshahi A, Javadi MR, Khoee SH, Alimoghaddam K, Ghavamzadeh A: The effect of zinc sulfate in the prevention of high-dose chemotherapy-induced mucositis: a double-blind, randomized, placebo-controlled study. Hematol Oncol 2012, 30(1):22–26. [DOI] [PubMed] [Google Scholar]
  • 23.Yarom N, Ariyawardana A, Hovan A, Barasch A, Jarvis V, Jensen SB, Zadik Y, Elad S, Bowen J, Lalla RV et al. : Systematic review of natural agents for the management of oral mucositis in cancer patients. Support Care Cancer 2013, 21(11):3209–3221. [DOI] [PubMed] [Google Scholar]
  • 24.Prasad AS, Beck FW, Snell DC, Kucuk O: Zinc in cancer prevention. Nutr Cancer 2009, 61(6): 879–887. [DOI] [PubMed] [Google Scholar]
  • 25.Choi S, Cui C, Luo Y, Kim SH, Ko JK, Huo X, Ma J, Fu LW, Souza RF, Korichneva I et al. : Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1. FASEB J 2018, 32(1):404–416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Sun D, Zhang L, Wang Y, Wang X, Hu X, Cui FA, Kong F: Regulation of zinc transporters by dietary zinc supplement in breast cancer. Mol Biol Rep 2007, 34(4):241–247. [DOI] [PubMed] [Google Scholar]
  • 27.Costello LC, Franklin RB, Feng P, Tan M: Re: Zinc supplement use and risk of prostate cancer. J Natl Cancer Inst 2004, 96(3):239–240; author reply 240-231. [DOI] [PubMed] [Google Scholar]
  • 28.He G, Zhu H, Yao Y, Chai H, Wang Y, Zhao W, Fu S, Wang Y: Cysteine-rich intestinal protein 1 silencing alleviates the migration and invasive capability enhancement induced by excessive zinc supplementation in colorectal cancer cells. Am J Transl Res 2019, 11(6):3578–3588. [PMC free article] [PubMed] [Google Scholar]
  • 29.Ninsontia C, Phiboonchaiyanan PP, Chanvorachote P: Zinc induces epithelial to mesenchymal transition in human lung cancer H460 cells via superoxide anion-dependent mechanism. Cancer Cell Int 2016,16:48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Bi Y, Lin GX, Millecchia L, Ma Q: Superinduction of metallothionein I by inhibition of protein synthesis: role of a labile repressor in MTF-1 mediated gene transcription. J Biochem Mol Toxicol 2006, 20(2):57–68. [DOI] [PubMed] [Google Scholar]
  • 31.Hardyman JE, Tyson J, Jackson KA, Aldridge C, Cockell SJ, Wakeling LA, Valentine RA, Ford D: Zinc sensing by metal-responsive transcription factor 1 (MTF1) controls metallothionein and ZnT1 expression to buffer the sensitivity of the transcriptome response to zinc.Metallomics 2016, 8(3):337–343. [DOI] [PubMed] [Google Scholar]
  • 32.Hogstrand C, Zheng D, Feeney G, Cunningham P, Kille P: Zinc-controlled gene expression by metal-regulatory transcription factor 1 (MTF1) in a model vertebrate, the zebrafish. Biochem Soc Trans 2008, 36(Pt 6): 1252–1257. [DOI] [PubMed] [Google Scholar]
  • 33.Ji L, Zhao G, Zhang P, Huo W, Dong P, Watari H, Jia L, Pfeffer LM, Yue J, Zheng J: Knockout of MTF1 Inhibits the Epithelial to Mesenchymal Transition in Ovarian Cancer Cells. J Cancer 2018, 9(24):4578–4585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Hedberg KK, Birrell GB, Mobley PL, Griffith OH: Transition metal chelator TPEN counteracts phorbol ester-induced actin cytoskeletal disruption in C6 rat glioma cells without inhibiting activation or translocation of protein kinase C. J Cell Physiol 1994, 158(2):337–346. [DOI] [PubMed] [Google Scholar]
  • 35.Zhu B, Wang J, Zhou F, Liu Y, Lai Y, Wang J, Chen X, Chen D, Luo L, Hua ZC: Zinc Depletion by TPEN Induces Apoptosis in Human Acute Promyelocytic NB4 Cells. Cell Physiol Biochem 2017, 42(5): 1822–1836. [DOI] [PubMed] [Google Scholar]
  • 36.Corniola RS, Tassabehji NM, Hare J, Sharma G, Levenson CW: Zinc deficiency impairs neuronal precursor cell proliferation and induces apoptosis via p53-mediated mechanisms. Brain Res 2008, 1237:52–61. [DOI] [PubMed] [Google Scholar]
  • 37.Donadelli M, Dalla Pozza E, Costanzo C, Scupoli MT, Scarpa A, Palmieri M: Zinc depletion efficiently inhibits pancreatic cancer cell growth by increasing the ratio of antiproliferative/proliferative genes. J Cell Biochem 2008, 104(1):202–212. [DOI] [PubMed] [Google Scholar]
  • 38.Mendivil-Perez M, Velez-Pardo C, Jimenez-Del-Rio M: TPEN induces apoptosis independently of zinc chelator activity in a model of acute lymphoblastic leukemia and ex vivo acute leukemia cells through oxidative stress and mitochondria caspase-3- and AIF-dependent pathways. Oxid Med Cell Longev 2012, 2012:313275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Rojas-Valencia L, Velez-Pardo C, Jimenez-Del-Rio M: Metal chelator TPEN selectively induces apoptosis in K562 cells through reactive oxygen species signaling mechanism: implications for chronic myeloid leukemia. Biometals 2017, 30(3):405–421. [DOI] [PubMed] [Google Scholar]
  • 40.Hongxia L, Yuxiao T, Zhilei S, Yan S, Yicui Q, Jiamin S, Xin X, Jianxin Y, Fengfeng M, Hui S: Zinc inhibited LPS-induced inflammatory responses by upregulating A20 expression in microglia BV2 cells. J Affect Disord 2019, 249:136–142. [DOI] [PubMed] [Google Scholar]
  • 41.Schaefer-Ramadan S, Barlog M, Roach J, Al-Hashimi M, Bazzi HS, Machaca K: Synthesis of TPEN variants to improve cancer cells selective killing capacity. Bioorg Chem 2019, 87:366–372. [DOI] [PubMed] [Google Scholar]
  • 42.Zhao G, Wang Q, Gu Q, Qiang W, Wei JJ, Dong P, Watari H, Li W, Yue J: Lentiviral CRISPR/Cas9 nickase vector mediated BIRC5 editing inhibits epithelial to mesenchymal transition in ovarian cancer cells. Oncotarget 2017, 8(55):94666–94680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Zhao G, Wang Q, Wu Z, Tian X, Yan H, Wang B, Dong P, Watari H, Pfeffer LM, Guo Y et al. : Ovarian Primary and Metastatic Tumors Suppressed by Survivin Knockout or a Novel Survivin Inhibitor. Mol Cancer Ther 2019, 18(12):2233–2245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Jarrard DF: Does zinc supplementation increase the risk of prostate cancer? Arch Ophthalmol 2005, 123(1): 102–103. [DOI] [PubMed] [Google Scholar]
  • 45.Sangthawan D, Phungrassami T, Sinkitjarurnchai W: Effects of zinc sulfate supplementation on cell-mediated immune response in head and neck cancer patients treated with radiation therapy. Nutr Cancer 2015, 67(3):449–456. [DOI] [PubMed] [Google Scholar]
  • 46.Sangthawan D, Phungrassami T, Sinkitjarurnchai W: A randomized double-blind, placebo-controlled trial of zinc sulfate supplementation for alleviation of radiation-induced oral mucositis and pharyngitis in head and neck cancer patients. J Med Assoc Thai 2013, 96(1):69–76. [PubMed] [Google Scholar]
  • 47.Lin YS, Lin LC, Lin SW: Effects of zinc supplementation on the survival of patients who received concomitant chemotherapy and radiotherapy for advanced nasopharyngeal carcinoma: follow-up of a double-blind randomized study with subgroup analysis. Laryngoscope 2009, 119(7): 1348–1352. [DOI] [PubMed] [Google Scholar]
  • 48.Kocdor H, Ates H, Aydin S, Cehreli R, Soyarat F, Kemanli P, Harmanci D, Cengiz H, Kocdor MA: Zinc supplementation induces apoptosis and enhances antitumor efficacy of docetaxel in non-small-cell lung cancer. Drug Des Devel Ther 2015, 9:3899–3909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Guerrerio AL, Berg JM: Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1). Biochemistry 2004, 43(18):5437–5444. [DOI] [PubMed] [Google Scholar]
  • 50.Auf der Maur A, Belser T, Elgar G, Georgiev O, Schaffner W: Characterization of the transcription factor MTF-1 from the Japanese pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal stress response. Biol Chem 1999, 380(2): 175–185. [DOI] [PubMed] [Google Scholar]
  • 51.Gefeller EM, Bondzio A, Aschenbach JR, Martens H, Einspanier R, Scharfen F, Zentek J, Pieper R, Lodemann U: Regulation of intracellular Zn homeostasis in two intestinal epithelial cell models at various maturation time points. J Physiol Sci 2015, 65(4):317–328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Burian Z, Ladanyi A, Barbai T, Piurko V, Garay T, Raso E, Timar J: Selective Inhibition of HIF1alpha Expression by ZnSO4 Has Antitumoral Effects in Human Melanoma. Pathol Oncol Res 2019. [DOI] [PubMed] [Google Scholar]
  • 53.Ostrakhovitch EA, Olsson PE, von Hofsten J, Cherian MG: P53 mediated regulation of metallothionein transcription in breast cancer cells. J Cell Biochem 2007, 102(6): 1571–1583. [DOI] [PubMed] [Google Scholar]
  • 54.Laskaris P, Atrouni A, Calera JA, d'Enfert C, Munier-Lehmann H, Cavaillon JM, Latge JP, Ibrahim-Granet O: Administration of Zinc Chelators Improves Survival of Mice Infected with Aspergillus fumigatus both in Monotherapy and in Combination with Caspofungin. Antimicrob Agents Chemother 2016, 60(10): 5631–5639. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1628086-supplement-1.pdf (633KB, pdf)

RESOURCES