Metallothionein 1X is a tumor suppressor gene and inhibits oxidative stress and metastasis in renal cell carcinoma

Abstract

Metallothioneins 1X (MT1X) is expressed at low levels in renal cell carcinoma (RCC) and correlates with tumor progression, stage, grade and prognosis, but the mechanism of MT1X’s role in renal cell carcinoma is not fully understood at present, and the aim of this study was to investigate the molecular mechanism of MT1X’s role in renal cell carcinoma. We used immunofluorescence and flow cytometry to detect intracellular reactive oxygen species (ROS) levels and immunoblotting to detect the expression levels of key proteins of the epithelial-mesenchymal transition (EMT) signaling pathway, transwell assay to assess the cell migration and invasion capacity. It was found that MT1X knockdown significantly upregulated H₂O₂-induced intracellular ROS, activated the EMT pathway, and ultimately promoted cell migration and invasion whereas Trolox inhibited cell migration and invasion by suppressing the elevated ROS induced by MT1X knockdown. Here, we reported that MT1X is low-expressed in RCC and that MT1X knockdown promotes cell migration and invasion through the upregulation of intracellular ROS levels, thereby activating the EMT pathway.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-025-02949-7.

Introduction

Renal cell carcinoma (RCC) is the second leading cause of death in human urological malignancies. Approximately 20–30% of RCC patients had metastatic lesions detected at initial diagnosis [1]. Clear cell Renal Cell Carcinoma (ccRCC) is the most important subtype of renal carcinoma, accounting for about 70% of renal carcinoma [2]. Although the 5-year overall survival rates of metastatic RCC have shown some improvement, the prognosis is still poor [3]. Currently, targeted agents or combination of targeted agents and immune-checkpoint inhibitors have been widely recommended as the first-line or second-line therapies for metastatic RCC, which have been shown to improve overall survival and prognosis. However, the drug resistance and toxic effects still limited treatment tolerability. Meanwhile, the molecular mechanisms of metastatic RCC have not been clearly elucidated.

Metallothioneins (MTs) are small cysteine-rich proteins that play an important role in regulating metal homeostasis and preventing heavy metal toxicity, DNA damage and oxidative stress [4, 5]. In mammals, four different MT-1 to MT-4 isoforms have been identified [6]. Metallothioneins I and II are wildly expressed in various tissues throughout the body, while MT3 is brain specific and MT4 is mainly expressed in skin and tounge [7–9]. Metallothioneins have an exceptionally high sulfur content because every of them contains more than 20 cysteine residues, granting them a high affinity for heavy metal ions (e.g., Zn, Cu, Cd) in order for them to participate in the physiological processes that protect cells against oxidative stress [10]. Zn could help superoxide dismutase 1 (SOD1) catalysis O2- to form O2 and H₂O₂, a vital physiological process to reduce cellular ROS [11]. Some of MT family members have been reported to be significantly altered in the expression level of various tumors and regulate tumor metastasis and progression [12, 13]. However, there are few studies on MT1X in renal cell carcinoma. We found that MT1X is lowly expressed in RCC, nevertheless RCC patients with low MT1X expression have a better prognosis, which is a paradoxical but interesting phenomenon. Therefore, this study is aimed at the function and significance of MT1X expression in RCC.

Despite significant progress in the diagnosis and therapy of cancer, metastasis is a major obstacle to improving clinical outcomes, as more than 90% of cancer-related deaths are the result of metastatic disease. Epithelial-mesenchymal transition (EMT), which is an evolutionarily conserved developmental process that contributes to the processes of histogenesis and organogenesis [14], might help facilitate researchers’ deeper understanding of the molecular mechanisms of tumor metastasis. The effects of molecular perturbations in EMT-inducible genes on metastasis have been widely explored [15]. Alteration of some key molecules in EMT might contribute to loss of specific cell-cell interactions in polarized epithelial cells, including tight junctions, gap junctions, cadherin based adherent junctions, and instead give epithelial cells some of the properties of mesenchymal cells, rendering them more susceptible to metastasis [16].

ROS, which are mainly generated as byproducts of mitochondrial respiratory chain and active NADPH oxidases (NOXs), are regulated by a series of redox reactions in biological systems and act as signaling molecules to drive cellular regulatory pathways [17]. It reported that ROS could induce DNA damage, leading to unrepaired or misrepaired DNA and subsequent tumor mutations [18]. Zhou et al. [19] reported that hypoxia induces the EMT signaling pathway in alveolar epithelial cells through activation of mitochondrial ROS, HIF, and endogenous TGF-β1 signaling. A large body of literature discusses the role of ROS in promoting cancer development and metastasis [20], indicating a vital role played by ROS in tumorigenesis.

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known antioxidant which is an analogue of vitamin E lacking the phytyl tail, could scavenge cellular peroxyl and alkoxyl radicals. Due to its increased cell permeability, Trolox exhibits an enhanced antioxidant capacity [21]. Numerous studies have shown that Trolox could slow down lipid peroxidation and reduce oxidative stress to decrease apoptotic cell death [22]. As a ROS scavenger [23], Trolox could inhibit aberrant oxidative stress in tumor cells. The aim of this study was to explore the clinical significance and molecular mechanism of MT1X in the metastatic progression of RCC.

Materials and methods

Patients and clinical data

From January 2007 to December 2020, 60 patients with RCC who underwent radical nephrectomy without preoperative treatment were enrolled in this study. The study was approved by the Biomedical Research Ethics Committee of Peking University First Hospital, and written informed consent was obtained from all participants. According to the TNM classification of malignant tumors by the Union for International Cancer Control (2011), all tissue samples were pathologically confirmed to be RCC. Nuclear grading was determined using the Foreman nuclear grading system.

Cell culture

The kidney renal clear cell carcinoma (KIRC) cell lines (786O) and human embryonic kidney cell line (HEK-293T) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The HEK293T cells were cultured in DMEM (Gibco), while 786O cells were cultured in PRMI1640(Gibco). All media were supplemented with 10% fetal bovine serum (Corning, Corning, NY) and 1% penicillin-streptomycin (Sigma-Aldrich). All cells were incubated at 37 °C in a humidified 5% CO2 atmosphere. The authenticity of all cell lines was verified using short tandem repeat analysis, and they were regularly tested for mycoplasma contamination.

Immunohistochemistry

Paraffin-embedded tissue Sect. (5 μm) were deparaffinized in xylene and rehydrated with graded concentrations of ethanol. Endogenous peroxidase activity was blocked using 0.35% H₂O₂. Antigen retrieval was performed by boiling the sections in 0.01 M citrate buffer (pH 6.0). Subsequently, sections were treated with 1% bovine serum albumin at 37 °C for 30 min to block nonspecific protein binding. Sections were then incubated with anti-MT1X antibody (Proteintech, 17172-1-AP) at 4 °C overnight. After incubation, they were treated with a biotinylated secondary antibody for 1 h at room temperature and stained using a 3,3-diaminobenzidine tetrahydrochloride substrate kit (Zhongshan Jinqiao Biotechnology, Beijing, China).

Lentivirus vector construction and cell transfection

Short hairpin RNA (shRNA) oligonucleotides targeting MT1X (sh-MT1X) and negative control shRNA (sh-Scramble) were designed and inserted into the pLKO.1 vector (Addgene, Watertown, MA). The full-length human MT1X complementary DNA was generated by PCR and cloned into the pLVX vector (Addgene, Watertown, MA, USA). The stable cell line was established by lentivirus infection accordingly. Lentiviral particles were produced using a three-vector system: transfer vector, viral packaging (ps·PAX2) and viral envelope (pMD2.G) at 4:3:1 ratio transfected into human 293 T cells. Then, the KIRC cells were infected by lentiviruses according to the MOI value (the number of lentiviruses per number of cells).

Quantitative RT-PCR

Total RNA was extracted from KIRC cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA was generated using the Reverse Transcription System (TansGEN, Beijing, China) according to the manufacturer’s instructions. RT-qPCR was performed in the 7500 Fast Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Inc) using SYBR Premix Ex Taq II (TaKaRa, Japan) according to the manufacturer’s instructions. Results were normalized to the endogenous control β-actin.

Cell metastasis assays

The migration and invasive abilities of KIRC cells were determined by wound-healing assay and transwell assay, respectively. For wound healing assays, cells were seeded into six-well plates at a density of 100,000/well and cultured in complete medium until the cells were nearly to fusion and then scraped in the wells with a sterilized 200 µl pipette tip. 0 h and 8 h later, cell migration in the wound area was visualized under a phase-contrast microscope. Overall migration distance was assessed using the ImageJ program (National Institutes of Health, Bethesda, MD).

The Transwell migration assays were performed using Transwell chambers (24-well inserts, 8 μm pore size, Corning Incorporated) Next, serum-free medium was used to inoculate 4 × 10⁴ cells in the upper chamber, and medium with 10% fetal bovine serum was added to the lower chamber.

For invasion assays, serum-free medium was used to inoculate 6 × 10⁴ cells in the upper chamber coated by the upper layer with Matrigel (product #354234, Corning Incorporated, New York, N.Y., diluted 1:8 in phosphate-buffered saline (PBS)), and incubated for 30 min at 37 °C for preparation. The culture conditions were the same as those described for the Transwell migration assay.

After 5 h of incubation, cells remaining in the upper chamber of the migration and invasion assays were wiped clean, and cells that had invaded to the bottom surface were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet diluted in methanol for 30 min. The cells in the lower lumen were observed under a phase contrast microscope (Leica DM IL, Leica Microsystems). The invasive cell counts were calculated by ImageJ software, and all experiments were performed in triplicate and repeated three times.

Animal experiments

In the tail-vein lung metastasis assay, 1 × 10⁶ stably transfected 786O-luc⁺-shMT1X-Scramble and 786O-luc⁺-shMT1X cells were injected into the tail vein of 5-week-old male NTG mice. Two weeks after injection, mice were anesthetized by isoflurane (Ichiban Pharmaceuticals) and injected intraperitoneally with d-fluorescein sodium salt (Biovision). Metastatic regions were detected in an in vivo imaging system Xenogen IVIS (PerkinElmer, MA) on average once a week. At the end of the experiment, all animals were sacrificed by carbon dioxide anesthesia. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Peking University First Hospital (Beijing, China).

Mitochondrial ROS assay

KIRC cells were treated with medium containing H₂O₂ for 4 h and stained with 5 µM DHE (KeyGEN, KGA7502-5, China) for 30 min to detect ROS generation. The stained cells were then analyzed with a fluorescence microscope (Olympus, Japan) or determined using flow cytometry.

Construction of ROSscore signature and statistical analysis

ROS related genes were obtained from the previous publication, and log2(TPM + 1) values of these genes were used to construct the ROS prognostic model in TCGA-KIRC. One-way COX regression analysis and P < 0.05 were used to screen prognostic genes. The genes related to all of overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFS) were subsequently analyzed by LASSO regression to contribute ROS prognostic model. The ROSscore was established via weighting the log2(TPM + 1) value of each key gene with the LASSO regression coefficient (“βi” represents LASSO regression coefficient and (“χi” represents log2(TPM + 1) value) [2]: ROSscore=∑βi × χi.

Immunoblot assay

Total protein extracts from cultured cells were separated using 8–12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). For MT1X with only 10kd, transfer for 20 min using a PVDF membrane with a pore size of 0.2 μm. After blocking with TBS-Tween buffer containing 5% skimmed milk at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4 °C. The membranes were then incubated with matched horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using the ECL Plus kit (Applygen Technologies Inc., Beijing, China). The blotting membranes were probed using antibodies against N-Cadherin (Cell Signaling Technology, #13116, Massachusetts, USA), E-Cadherin (Cell Signaling Technology, #9782, Massachusetts, USA), β-Catenin (Cell Signaling Technology, #8480, Massachusetts, USA), Vimentin (Cell Signaling Technology, #5741, Massachusetts, USA) and β-Tubulin (Zhongshan Jinqiao Biotechnology, Beijing, China). To improve clarity and focus on relevant results, certain Western blot images were cropped to display only the bands corresponding to the target proteins.

Statistical analysis

The Student’s t-test and Kruskal-Wallis test were used for raw data analysis. Survival analysis was performed using the Kaplan-Meier method and log-rank test. Prognostic correlations between clinicopathologic and immunohistochemical data were assessed by univariate and multivariate Cox regression analysis. SPSS version 22.0 software (SPSS, Chicago, IL) was used for statistical analysis. All values in the text and graphs are expressed as the mean ± standard deviation (SD) of these observations. Statistical significance was considered at P-value < 0.05 (*P < 0.05, **P < 0.01 and ***P < 0.001).

Results

MT1X is downregulated in RCC but correlates with poor outcomes in RCC patients

Analysis of the TCGA database showed that MT1X mRNA expression was reduced in KIRC tissues compared to normal tissues (Fig. 1A). A comparison of paired samples showed that tumor tissues exhibited higher MT1X expression levels compared to adjacent normal tissues (Fig. 1B). Further analysis revealed that high MT1X mRNA expression was associated with advanced tumor pathological stage (Fig. 1C, D) (p < 0.01). Next, the correlation of patients’ OS, DSS and progression-free interval (PFI) with MT1X expression levels in the TCGA-KIRC database was assessed by Kaplan-Meier survival analysis and log-rank test. However, the results showed that patients with high MT1X expression had worse OS (p < 0.001), DSS (p < 0.001) and PFI (p < 0.001) compared to patients with low MT1X expression (Fig. 1E–G).

Fig. 1.

High expression of MT1X is associated with poor prognosis in KIRC. A Analysis of the relative expression of MT1X mRNA in KIRC tissues compared to normal tissues and paired samples B in the TCGA database. C Correlation between MT1X and pathological T-stage and M-stage (D) of KIRC. MT1X expression was significantly associated with (E) overall survival, F disease-specific survival and (G) progression-free period in the TCGA-KIRC cohort according to Kaplan-Meier analysis and log-rank tests. H Representative immunostaining pictures of MT1X expression in different KIRC pathological stage tissues and paired paraneoplastic renal tissue. Scale bar, 100 μm and 50 μm, respectively. I Immunohistochemistry staining analysis of MT1X protein expression in 59 RCC tissues and 51 adjacent normal kidney tissues in IUPU cohort. J Immunohistochemistry staining analysis of MT1X protein expression in 21 T1, 20 T2 and 19 T3 RCC tissues in IUPU cohort. TCGA, The Cancer Genome Atlas; KIRC: Kidney renal clear cell carcinoma. *p < 0.05, **p < 0.01, ***p < 0.001

To validate the clinical significance of MT1X, immunohistochemistry (IHC) analysis was conducted to assess MT1X expression levels in KIRC patients. The results showed that histochemical staining was generally lower in KIRC tissues compared to normal adjoining tissues, but IHC staining intensity increased with elevated pathologic T-stage (Fig. 1H). We next scored the intensity of IHC staining in a RCC cohort at our center (Institute of Urology, Peking University), including 59 RCC and 51 paired adjacent normal tissues (Fig. 1I), and the results were analyzed by different T-stages (Fig. 1J). We found that MT1X expression was indeed lower in RCC than in adjacent normal tissues, however, IHC scores tended to increase with elevated T-stage and were of statistical significance, which was consistent with the expression pattern in the TCGA database. Collectively, the above results suggest that MT1X is downregulated in KIRC tissues, but as the pathological stage of the tumor rises, so does the expression of MT1X, although it remains lower compared to normal tissues. Paradoxically, despite the low expression of MT1X in KIRC, patients with high MT1X expression of KIRC have a worse prognosis.

MT1X knockdown promotes migration and invasion capacity of RCC cell lines

Considering the contradictory MT1X expression pattern and its association with prognosis in KIRC, to investigate the mechanism of MT1X function in RCC malignancies, we conducted lentivirus-mediated knockdown of MT1X in RCC cell line 786O, using a nonsense scramble sequence as a negative control. Knockdown efficiency was assessed by quantitative RT-PCR (Fig. 2A) and immunoblot assays (Fig. 2B). Considering the deleterious effect of tumor metastasis on survival rates, we evaluated the ability of MT1X to promote RCC metastasis. The migration and invasion abilities of tumor cells were evaluated using the matrix transwell assay and the wound healing assay. MT1X knockdown significantly enhanced tumor cell migration and invasion compared with control scramble (Fig. 2C). In contrast, MT1X overexpression inhibited tumor cell migration and invasion (Fig. 2D). The MT1X knockdown group also showed enhanced migration in the wound healing assay (Figure S1A and S1C), which could be rescued by MT1X overexpression (Figure S1B and S1D). Together, these findings suggest that MT1X may function as a tumor suppressor gene in RCC which inhibits the RCC cell migration and invasion ability.

Fig. 2.

MT1X promotes RCC cell migration and invasion in vitro and in vivo. A The MT1X knockdown efficiency in 786O was assessed by RT-qPCR. B Protein levels of MT1X after MT1X knockdown in 786O cells were detected by immunoblot assays. C MT1X knockdown enhanced cell migration and invasion compared to scramble. D Overexpression of MT1X reduced tumor cell migration and invasion. E–H Statistical results of relative migration/invasion count. I Two weeks after injection, metastatic regions were detected in an in vivo imaging system Xenogen IVIS. B Representative IVIS images of mice injected 786O-luc⁺-shMT1X-Scramble or 786O-luc⁺-shMT1X cells. J Curve of photon flux of lung metastatic tumors with time, n = 10. D Analysis of luminescence representing lung metastasis measured on day 28. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. SCR scramble, OE overexpression. Scale bar, 50 μm

To further explore the role of MT1X in KIRC metastasis, we established an in vivo tumor metastasis model by tail-vein injection and injected stably transfected luciferase-conjugated 786O-luc⁺-Scramble and 786O-luc⁺-shMT1X cells into NTG mice, respectively, to generate an animal model of renal cell carcinoma lung metastasis. The results showed that all mice developed lung metastases after 14 days (Fig. 2I), and the fluorescence intensity of lung metastases was higher in mice injected with 786O-shMT1X-#1 cells compared to mice injected with 786O-Scramble cells at day 28 (Fig. 2J), indicating that MT1X knockdown KIRC cells have a higher metastatic capacity in vivo.

MT1X decreases H₂O₂-induced increasing ROS in RCC cell lines

Since MT1X is cysteine-rich and localized in the mitochondrial membrane, it is able to bind to intracellular heavy metal ions, which is closely related to oxidative stress. ROS are the major and significant products of oxidative stres, which play important role in tumorigenesis, development and metastasis. Considering the above factors, we evaluated the effect of MT1X on intracellular ROS in RCC cells, aiming to further elucidate the mechanism of MT1X to suppress RCC metastasis. We treated the cells with the strong oxidant H₂O₂ intended to induce an increase in intracellular ROS. Next, we co-cultured cells with dihydroethidium (DHE), a peroxide indicator to visualize changes in intracellular ROS. Under fluorescence microscopy, it was found that 500 µM H₂O₂ treatment for 4 h significantly increased intracellular ROS levels, which was more pronounced in the MT1X knockdown group (Fig. 3A), while ROS elevation was appropriately suppressed in the MT1X overexpression group (Fig. 3B). Flow cytometry assay also showed that 500 µM H₂O₂ treatment promoted ROS generation in shMT1X-#1 and shMT1X-#2 versus shMT1X-#SCR (Fig. 3C), while ROS elevation was also inhibited in the MT1X overexpression group (Fig. 3D). By comparison of the levels of endogenous ROS under H₂O₂ treatment (Fig. 3E, F), we found that H₂O₂ significantly induced an elevation of endogenous ROS in MT1X knockdown cells (Fig. 3E). And despite the relatively weak effect, overexpression of MT1X was able to inhibit H₂O₂-induced endogenous ROS in cells to some extent (Fig. 3F). Summarizing these results, we hypothesized that MT1X was able to inhibit intracellular oxidative stress events when external oxidative stimuli existed.

Fig. 3.

MT1X decreases H₂O₂-induced intercellular ROS. Using DHE as a ROS indicator. A MT1X knockdown enhanced H₂O₂-induced intercellular ROS compared to scramble. B Overexpression of MT1X reduced H₂O₂-induced intercellular ROS. C The endogenous ROS levels in shMT1X-Scramble, shMT1X-#1 and shMT1X-#2 were measured by flow cytometry. D The endogenous ROS levels in vector or MT1X-OE were measured by flow cytometry. Scale bar, 50 μm. DHE, dihydroethidium; ROS, reactive oxygen species; OE, overexpression. E Comparison of endogenous ROS levels of shMT1X-SCR, shMT1X-#1 and shMT1X-#2 under H₂O₂ treatment. F Comparison of endogenous ROS levels of vector and MT1X-OE under H₂O₂ treatment

Association of ROS with KIRC prognosis

Since MT1X inhibits H₂O₂-induced ROS upregulation in KIRC cells, it is necessary to explore ROS and the prognosis of KIRC tumors. Therefore, we constructed ROSscore-based prognostic models (Fig. 4A, B). Leone et al. [24] identified a tumor type-specific oxidative stress gene profile containing 69 genes by combining publicly available oxidative stress gene signatures with patient survival data from breast, lung, HNSCC, pancreatic, prostate, and colon cancers in the TCGA. Then, log2(TPM + 1) values of these genes were used to construct the ROS prognostic model in TCGA-KIRC. One-way COX regression analysis and P < 0.05 were used to screen prognostic genes. The genes related to all of OS, DSS and PFS were subsequently analyzed by LASSO regression to contribute ROS prognostic model. By analyzing gene expression in TCGA-KIRC patients, we found that MT1X expression was positively correlated with ROSscore (Fig. 4C), and as expected, KIRC patients with a high ROSscore had a poor prognosis for OS, DSS, and PFI (Fig. 4D–F).

Fig. 4.

Relationship of ROS signature score and prognosis in KIRC patients. A Study flowchart showing steps involved in construction of ROSscore-based prognostic signatures. B Elucidation for LASSO coefficient profiles of 26 prognostic oxidants and redox signaling gene for PFI. C MT1X expression was positively correlated with ROS signature score. ROSscore was significantly associated with (D) overall survival, (E) disease-specific survival and (F) progression-free interval in the TCGA-KIRC cohort according to Kaplan-Meier analysis. Forest plot showed the association between clinical parameters, MT1X expression, ROSscore and OS (G), DSS (H) and PFI (I) using multivariate analyses. Samples were divided into high- and low- score groups according to the median values. *p < 0.05, **p < 0.01, ***p < 0.001

Since MT1X inhibits oxidative stress, migration and invasion of KIRC cells and executes tumor suppressor functions, while paradoxically correlating with worse prognosis in patients with high MT1X expression (Fig. 1E–G), we performed a multivariate Cox regression analysis to further explore the relationship between MT1X and KIRC prognosis. The results showed that ROSscore was a high-risk factor for OS, DSS, and PFI in KIRC patients, whereas the expression level of MT1X was not significant (Fig. 4G–I).

Collectively, these results suggest that MT1X is highly correlated with ROSscore and that the reason why patients with high MT1X expression have a worse prognosis is that these patients have a higher ROSscore. Considering the anti-oxidative stress property of MT1X, the positive correlation between ROSscore and MT1X may be due to some negative feedback mechanism. We propose the assumption that MT1X in normal tissues exercises the function of protecting cells against oxidative stress, and in the early stage of tumors, low MT1X expression and impaired antioxidant capacity of tumors lead to the accumulation of endogenous ROS and an increasing of ROSscore, which further promotes the tumor progression and the elevation of T-stage; whereas in the late stage of tumors, the accumulated high ROSscore activates cellular self-protection mechanisms, which in a negative feedback manner causing the elevation of MT1X.

There are many other genes with inconsistent function and prognosis similar to MT1X, for example, nuclear factor (erythroid-derived-2)-like 2 (NFE2L2), also known as NRF2, has been traditionally considered as a tumor suppressor by regulating the transcription of downstream genes to activate cellular antioxidant responses to protect cells from exogenous and endogenous damage [25]. However, several literature reports that high expression of NEL2L2 in several cancer species indicates a poor prognosis [26, 27].

MT1X EMT pathways in KIRC cell lines

To clarify the mechanism by which MT1X inhibits cell migration, we detected EMT pathways which are closely related to tumor metastasis. Immunoblotting assays showed that the expression of N-Cadherin, β-Catenin and Vimentin in the EMT pathway was elevated in MT1X knockdown cell lines (Fig. 5A) whereas it was moderately decreased in the MT1X overexpressed cell lines (Fig. 5B). This suggests that MT1X negatively regulates the EMT pathways, consistent with its role in suppressing KIRC migration and invasion.

Fig. 5.

MT1X inhibits activation of epithelial-mesenchymal transition (EMT) pathways via decreasing intercellular ROS in 786O. A MT1X knockdown significantly activates key molecules like N-Cadherin, β-Catenin and Vimentin involved in EMT pathways, while MT1X overexpression inhibits expression of N-Cadherin, β-Catenin and Vimentin (B). C Treating cells with H₂O₂ produces a similar effect on the EMT pathway as MT1X knockdown, namely upregulation of N-Cadherin, β-Catenin and Vimentin. D Treating cells with the antioxidant Trolox inhibited activated N-Cadherin and Vimentin in MT1X knockdown cells (Left). Statistical results of the intensity of the protein bands of N-Cadherin and Vimentin/β-Tubulin relative to the shMT1X-SCR cells (Right) Data are shown as mean ± SD. *p < 0.05, **p < 0.01. SCR scramble

Trolox suppresses the EMT pathways activated by MT1X knockdown in KIRC cell lines

As MT1X is closely related to ROS, we wondered whether the regulation of the EMT pathway by MT1X is related to ROS, therefore we treated KIRC cells with 500 µM H₂O₂, and noticed that H₂O₂ could also arouse expression of N-Cadherin, β-Catenin and Vimentin (Fig. 5C), suggesting that ROS might activate the EMT pathway. To further investigate whether MT1X knockdown promotes KIRC cell migration and invasive function by activating the EMT pathway through upregulation of endogenous ROS, we treated MT1X knockdown cells with an antioxidant, Trolox, a vitamin E analog that scavenges intracellular ROS. We found that despite the upregulation of N-Cadherin and Vimentin in MT1X knockdown cells, N-Cadherin and Vimentin were moderately restored after treatment with 200 μm Trolox for 24 h (Fig. 5D), demonstrating that activation of the EMT pathway by MT1X knockdown is at least partially dependent on upregulation of endogenous ROS.

Trolox inhibits migration and invasion in MT1X knockdown KIRC cell lines

Previous immunoblot analysis showed that 200 μm Trolox treatment inhibited N-Cadherin and Vimentin expression in the EMT pathway in 786O cells (Fig. 5D). To further confirm how Trolox regulates cell function, we treated scramble or MT1X knockdown KIRC cells with Trolox for 24 h, respectively. We found that, compared to negative control group, Trolox treatment inhibited MT1X knockdown-induced cell migration (Fig. 6A) and invasion (Fig. 6B). This finding is consistent with the immunoblot result (Fig. 5D), suggesting that Trolox inhibits EMT pathways via its ROS scavenging capacity to suppress MT1X knockdown induced RCC cell migration and invasion.

Fig. 6.

Trolox suppresses migration and invasion of MT1X knockdown cells. Treating cells with 200 μm Trolox resulted in inhibition of cell migration (A) and invasion (B) compared to the negative control, which was more pronounced in MT1X knockdown cells. C, D Statistics of relative migrating/invading cell counts. Data are shown as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001

Discussion

MTs bind to physiological heavy metals, regulate intracellular zinc/copper balance, which is essential for cell proliferation and differentiation, and function as antioxidants to protect cells from free radicals and oxidative stress induced by air pollutants, mutagens, chemotherapeutic agents and radiation [10]. Given the critical role of MTs in physiological and pathological processes, a growing body of evidence suggests their potential as molecular targets for the treatment of human diseases, including neurodegenerative disorders, cerebral ischemia, retinal disorders, liver disorders, chemical and radiation-induced carcinogenesis, lung inflammation and obesity [28]. Our finding shows that MT1X functions as a tumor suppressor gene in KIRC and inhibits cell migration and invasion by suppressing oxidative stress in KIRC cells. The mRNA expression levels in the TCGA database showed that MT1X was low expressed in KIRC compared to adjacent normal tissue (Fig. 1A, B). In contrast, at higher pathological T and M stages, MT1X expression was elevated, and patients with high MT1X expression had a poorer prognosis.

Since MT1X inhibits oxidative stress in KIRC cells (Fig. 3), we constructed a ROS signature model (Fig. 4A) and found that the expression level of MT1X was positively correlated with ROSscore (Fig. 4C). Multivariate Cox regression analysis showed that ROSscore was a risk factor for OS, DSS, and PFI in KIRC patients, whereas the expression level of MT1X was not significantly associated (Fig. 4G–I). The above results indicate that MT1X is not likely to contribute to poor prognosis. Given that MT1X is positively correlated with ROSscore, patients with high MT1X expression tend to have elevated ROSscore, which is likely a significant factor contributing to poor prognosis. The reason MT1X inhibits oxidative stress but is positively correlated with ROSscore may be due to certain intracellular negative feedback regulatory mechanisms that lead to the upregulation of the tumor suppressor and antioxidant gene MT1X. Further IHC staining result shows that MT1X is expressed at lower levels in KIRC tissue compared to adjacent normal tissue, indicating that MT1X is aberrantly downregulated in KIRC. Previous studies have reported that MT1G inhibits cancer cell growth in pancreatic cancer [29] and thyroid cancer [30], and MT2A inhibits lung cancer cell migration [31]. However, the role of the MTs family in renal cell carcinoma remains underexplored. Our study is the first to report that MT1X functions as a tumor suppressor in cell renal cell carcinoma.

The well-known property of MTs is that they are localized in the mitochondrial membrane, binding to physiological heavy metals and regulate cellular homeostasis. Given the wide range of functions of MTs and the large number of members of the MTs family, the specific roles of individual isoforms have not yet been fully investigated. MT2A has been reported as an antioxidant, playing an important role in cardiovascular disease, neurological disorders, and cancer [32]. It has been reported that E2F transcription factor 4 promotes autophagy in a cell cycle-dependent manner, leading to accelerated MT1E, MT1M, and MT1X protein degradation, elevated Zn²⁺ distribution within the autophagosome, and a decrease in intracellular unstable zinc ions, which increase the growth, invasion, and metastasis of gastric cancer cells [13], suggesting a tumor suppressing mechanism of MT1X in gastric cancer. However, the mechanism by which MT1X exerts its action in renal cell carcinoma is currently unknown. Gain- and loss-of-function studies have demonstrated that MT1X inhibits KIRC cell migration and invasion (Fig. 2). In vivo tail-vein injection experiments showed that MT1X knockdown promotes lung metastasis (Fig. 2). By detecting the levels of intracellular ROS with H₂O₂ administration, we found that MT1X could inhibit H₂O₂-induced oxidative stress (Fig. 3). EMT pathways are closely related to cell migration and invasion function, further results indicated that MT1X could regulate N-Cadherin, β-Catenin and Vimentin, which are key modulators in EMT pathways (Fig. 5A and B). Considering the strong association between MT1X and oxidative stress, we hypothesized that MT1X’s effect on the EMT pathway might be ROS-dependent. To test this, we evaluated the expression level of the EMT pathway after treating the cells with H₂O₂, a powerful oxidant, and found that H₂O₂ was able to upregulate N-Cadherin, β-Catenin and Vimentin in the EMT pathway (Fig. 5C), which was consistent with the results caused by MT1X knockdown (Fig. 5A). Since MT1X knockdown increases ROS (Fig. 3), we speculated that it might activate the EMT pathway via enhancing oxidative stress.

Vitamin E (Vit-E) is an important natural antioxidant, known to play a central role in the antioxidant defense response [33], exhibiting ROS scavenging function. The fat-soluble nature of Vit-E allows it to easily penetrate the phospholipid bilayer membrane, thereby inhibiting ferroptosis and lipid peroxidation [34]. However, the highly hydrophobic nature of vitamin E reduces its solubility in buffer solutions, so its antioxidant activity is not always consistent in experimental models. Trolox is a water-soluble derivative of vitamin E [35]. It was reported that Trolox reduces the migration and invasive activity of breast cancer 4T1 cells through a prostaglandin E2 (PGE2)-dependent and independent mechanism [36]. Besides, Trolox could inhibit NFκB-mediated MMP-9 expression, thereby suppressing migration and invasion of lung and cervical cancer cells [37]. Consistent with these results, in this study, we reported that Trolox inhibited cell migration and invasion induced by MT1X knockdown (Fig. 6). We also found that Trolox inhibited the upregulation of N-Cadherin and Vimentin in the EMT pathway induced by MT1X knockdown (Fig. 5D).

Conclusion

Our results confirmed that MT1X inhibited H₂O₂-induced ROS elevation. MT1X was able to regulate the expression of N-Cadherin, β-Catenin and Vimentin in the EMT pathway of renal cell carcinoma cell lines, thereby regulating cell migration and invasion. Trolox could block the generation of ROS in cells, and treating cells with Trolox downregulated the expression of N-Cadherin and Vimentin in the EMT pathway, thereby inhibiting cell migration and invasion (Figure S2). Briefly, in this study, we demonstrated that MT1X is a tumor suppressor gene in renal cell carcinoma, performs antioxidant functions, and inhibits tumor cell migration and invasion. Further work is needed to confirm the molecular mechanism of tumor metastasis inhibition by MT1X in renal cell carcinoma.

Author contributions

Conceptualization, S.H. and L.Z.; methodology, R.R.; software, C.H.; validation, C.H., Y.W. and H.G.; formal analysis, Y.W.; investigation, R.R.; resources, Y.G.; data curation, R.R.; writing—original draft preparation, R.R.; writing—review and editing, R.R.; visualization, H.G.; supervision, X.L.; project administration, S.H.; funding acquisition, L.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (81972379, 82173216, 81872083, and 81972380), the Capital Health Research and Development of Special Fund (2022-1-4072), the Clinical Features Research of Capital (Z211100002921070), Clinical Medicine Plus X—Young Scholars Project, Peking University, the Fundamental Research Funds for the Central Universities (PKU2021LCXQ026), and Wuxi “Taihu Talents Program” Medical and Health High-level Talents Project.

Data availability

All of the data are presented in this article.

Declarations

Ethics approval and consent for participate

This study was approved by the Medical Ethics Committee of Peking University First Hospital (2024-029) and conducted in accordance with the Declaration of Helsinki. Informed consent was waived due to the retrospective use of anonymized clinical samples. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Peking University First Hospital (Beijing, China).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.