metallothionein and Carcinoma--Renal-Cell

Clear-cell renal cell carcinoma (ccRCC) appears as the most common type of kidney cancer, the carcinogenesis of which has not been fully elucidated. Tumor heterogeneity plays a crucial role in cancer progression, which could be largely deciphered by the implement of scRNA-seq. The bulk and single-cell RNA expression profile is obtained from TCGA and study conducted by Young et al. We utilized UMAP, TSNE, and clustering algorithm Louvain for dimensionality reduction and FindAllMarkers function for determining the DEGs. Monocle2 was utilized to perform pseudo-time series analysis. SCENIC was implemented for transcription factor analysis of each cell subgroup. A series of WB, CFA, CCK-8, and EDU analysis was utilized for the validation of the role of MT2A in ccRCC carcinogenesis. We observed higher infiltration of T/NK and B cells in tumorous tissues, indicating the role of immune cells in ccRCC carcinogenesis. Transcription factor analysis revealed the activation of EOMES and ETS1 in CD8 + T cells, while CAFs were divided into myo-CAFs and i-CAFs, with i-CAFs showing distinct enrichment of ATF3, JUND, JUNB, EGR1, and XBP1. Through cell trajectory analysis, we discerned three distinct stages of cellular evolution, where State2 symbolizes normal renal tubular cells that underwent transitions into State1 and State3 as the CNV score ascended. Functional enrichment examination revealed an amplification of interferon gamma and inflammatory response pathways within tumor cells. The consensus clustering algorithm yielded two molecular subtypes, with cluster 2 being associated with advanced tumor stages and an abundance of infiltrated immune cells. We identified 17 prognostic genes through Cox and LASSO regression models and used them to construct a prognostic model, the efficacy of which was verified in multiple cohorts. Furthermore, we investigated the role of MT2A, one of our hub genes, in ccRCC carcinogenesis, and found it to regulate proliferation and migration of malignant cells. We depicted a detailed single-cell landscape of ccRCC, with special focus on CAFs, endothelial cells, and renal tubular cells. A prognostic model of high stability and accuracy was constructed based on the DEGs. MT2A was found to be actively implicated in ccRCC carcinogenesis, regulating proliferation and migration of the malignant cells.

Topics: Carcinogenesis; Carcinoma; Carcinoma, Renal Cell; Endothelial Cells; Humans; Kidney Neoplasms; Metallothionein; Single-Cell Gene Expression Analysis

The metallothionein 1 (MT1) family was previously shown to be involved in metal ion homeostasis, DNA damage, oxidative stress, and carcinogenesis. Our team's previous study showed that MT1X is most closely associated with ccRCC. However, its role in clear cell RCC (ccRCC) remains unclear. The present study aimed to demonstrate MT1X's prognostic value, potential biologic function, impact on the immune system, and influence on cell growth, the cell cycle, apoptosis, and migration in the setting of ccRCC. The relationship between clinical pathologic features and MT1X was analyzed using bioinformatics. We knocked down MT1X in the ccRCC cell line 786O with si-MT1X to verify the results of the bioinformatic analysis at the cytological level. Apoptosis assay, cell cycle assay, wound-healing assay, colony formation assay, and RT-qPCR were performed. MT1X is correlated with the stage (T and M) and grade and is able to be an independent prognostic factor for ccRCC. The TISIDB database analysis showed a significant correlation between MT1X and tumor-infiltrating lymphocytes such as central memory CD8+ T cells and γΔT cells. MT1X was also positively related to immunomodulators such as TGFB1 and CXCR4. We also found that MT1X knockdown inhibits cell growth, induces apoptosis, arrests cells in the S cell cycle, and inhibits the wound healing proportion in ccRCC. Gene set enrichment analysis and quantitative PCR (q-PCR) analysis found that down-regulation of MT1X reduced the accumulation of hypoxia-associated factors. Bioinformatic analysis associated increased MT1X expression with a worse prognosis. Laboratory experiments confirmed bioinformatic findings. MT1X was also found to be an independent prognostic biomarker for ccRCC and is involved in immune system regulation.

Topics: Biological Products; Biomarkers; Carcinoma, Renal Cell; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Metallothionein; Oncogenes

Metallothioneins are low‑weight cysteine‑rich proteins responsible for metal ion homeostasis in a cell and, thus, capable of regulating cell proliferation and differentiation. Deregulation of metallothionein genes has been reported in various human tumors. However, their role in renal cell carcinoma (RCC) has been poorly investigated. In the present study, we aimed to evaluate the importance of promoter DNA methylation of selected metallothionein genes for RCC. Based on the initial analysis of kidney renal clear cell carcinoma dataset from The Cancer Genome Atlas, genes MT1E, MT1F, MT1G and MT1M were selected for qualitative methylation analysis in 30 tumors (including 10 multifocal cases), 10 pericancerous, and 30 non‑cancerous renal tissues (NRT). Methylation of MT1E and MT1M was tumor‑specific (P=0.0056 and P=0.0486, respectively) and showed moderate interfocal variation in paired tumor foci. Methylated promoter status of the two genes was associated with larger tumor size (P=0.0110 and P=0.0156, respectively). Furthermore, aberrant MT1E methylation was more frequent in tumors having necrotic zones (P=0.0449) or characterized with higher differentiation grade (P=0.0144), while MT1M was more commonly methylated in tumors with higher Fuhrman grade (P=0.0272). Only unmethylated MT1F promoter status was observed in all analyzed samples. Gene expression analysis (51 RCC and 9 NRT) revealed MT1G downregulation in tumors (P<0.0001), while lower MT1E expression levels were associated with the promoter methylation (P=0.0077). In clear cell RCC, MT1E, MT1G and MT1M expression was higher than that noted in other histological tumor subtypes (all P<0.0500). In addition, some associations were observed between metabolic syndrome‑related clinical parameters and promoter methylation or gene expression. In conclusion, the present study revealed the potential role of MT1E and MT1M promoter methylation in RCC development.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Metallothionein; Middle Aged; Promoter Regions, Genetic

Functional epigenetic studies aimed to re-express transcriptionally silenced genes in renal cell carcinoma (RCC) may facilitate the ongoing search for appropriate markers supporting clinical decision-making.. The RCC cell line A-498 was treated with the DNA methyltransferase inhibitor zebularine under low-cytotoxicity conditions. RNA chip analyses revealed several upregulated transcripts that were further validated by qPCR on 49 matched pairs of human kidney tissues to identify suitable marker candidates.. Members of the metallothionein (MT) group were remarkably downregulated in tumor tissues. MT1G and MT1H expression was decreased in 98% of cases, whereas MT2A expression was downregulated in 73% of all cases. Comparison of 308 reactivated transcripts upregulated more than 1.5-fold to published data revealed a high number of shared candidates, which supports the consistency of this experimental approach.. MTs were found to be transcriptionally inactivated in human RCC. Our observations support the hypothesis of a possible involvement of these metalloproteins in renal cell carcinogenesis. Additional functional studies of these genes may provide clues for understanding renal cancers as essentially metabolic diseases.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Cytidine; DNA Modification Methylases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Metallothionein; Middle Aged; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcription, Genetic

We identified significantly hypermethylated genes in clear cell renal cell carcinoma.. We previously identified a set of under expressed genes in renal cell carcinoma tissue through transcriptional profiling and a robust computational screen. We selected 19 of these genes for hypermethylation analysis using a rigorous search for the best candidate regions, considering CpG islands and transcription factor binding sites. The genes were analyzed for hypermethylation in the DNA of 38 matched clear cell renal cell carcinoma and normal samples using matrix assisted laser desorption ionization time-of-flight mass spectrometry. The significance of hypermethylation was assessed using 3 statistical tests. We validated the down-regulation of significantly hypermethylated genes at the RNA and protein levels in a separate set of patients using reverse transcriptase-polymerase chain reaction, immunohistochemistry and Western blots.. We found 7 significantly hypermethylated regions from 6 down-regulated genes, including SFRP1, which was previously shown to be hypermethylated in renal cell carcinoma and other cancer types.. To our knowledge we report for the first time that another 5 genes (SCNN1B, SYT6, DACH1, and the tumor suppressors TFAP2A and MT1G) are hypermethylated in renal cell carcinoma. Robust computational screens and the high throughput methylation assay resulted in an enriched set of novel genes that are epigenetically altered in clear cell renal cell carcinoma. Overall the detection of hypermethylation in these highly down-regulated genes suggests that assaying for their methylation using cells from urine or blood could provide the basis for a viable diagnostic test.

Topics: Biomarkers, Tumor; Blotting, Western; Carcinoma, Renal Cell; CpG Islands; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Epithelial Sodium Channels; Eye Proteins; Humans; Intercellular Signaling Peptides and Proteins; Kidney Neoplasms; Membrane Proteins; Metallothionein; Reverse Transcriptase Polymerase Chain Reaction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Synaptotagmins; Transcription Factor AP-2; Transcription Factors

Good molecular markers for investigating the biochemical differences between renal cancer and surrounding tissues have not yet been developed. Sixteen kidney samples (clear cell RCC) were investigated to determine the differences in the protein components between renal cancer and surrounding tissues, using HPLC analysis. The metallothionein (MT) and zinc levels were consistently lower in renal cancer tissues compared with in surrounding tissues. The mean concentration of MT in normal tissues surrounding renal tumors was about 15 times higher than that in cancer tissues. An immunohistochemical study confirmed that the expression of MT in renal cancer tissues was lower than that in adjacent normal tissues. The activities of aminopeptidases (APs) were significantly decreased in renal cancer tissues compared with in adjacent normal tissues. An immunohistochemical study and Western blot analysis confirmed that the expression of AP-N in renal cancer tissues was also lower than in adjacent normal tissues. These results suggest that the immunohistochemical detection of MT and AP-N could provide useful information as a pathological diagnostic tool for classifying renal cancer and surrounding tissues.

Topics: Biomarkers, Tumor; Carcinoma, Renal Cell; CD13 Antigens; Chromatography, High Pressure Liquid; Humans; Immunohistochemistry; Kidney; Kidney Neoplasms; Metallothionein; Reference Values; Zinc

We investigated the immunohistochemical localization of metallothionein (MT) in renal cell carcinoma and determined the potential role of MT expression as a possible prognostic variable for tumor proliferation and progression.. Tumor tissue blocks from 70 patients with renal cell carcinoma who underwent radical or partial nephrectomy were investigated. Mean followup plus or minus standard error was 36 +/- 3 months. Immunohistochemical testing was performed by the avidin-streptavidin method using a monoclonal mouse antiMT antibody. MT staining intensity in samples was evaluated semiquantitatively. The subcellular distribution of MT was also determined. Staining characteristics were compared with the clinicopathological results.. MT immunostaining was found in 39 of 70 tumors (55.7%) and subcellulary MT was localized in the cytoplasm, nucleus and cell membrane. The survival of patients with MT immunostaining was significantly worse than that of those with MT negative results (p = 0.02). A significant relationship of higher tumor grade and MT staining intensity was observed in grades I and III (p = 0.01), and grades II and III (p = 0.02) tumors. No association was found of MT expression and pathological stage. Sarcomatoid tumors showed significantly higher MT expression than clear cell, papillary, granular or chromophobe tumors (p = 0.02, 0.001, 0.01 and 0.01, respectively). MT expression was not an independent prognostic variable.. MT over expression seems to be associated with malignant behavior and poor prognosis in renal cell carcinoma. Therefore, MT expression may be considered a useful marker of less differentiated and more aggressive renal cell carcinoma.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Renal Cell; Cell Membrane; Cell Nucleus; Cytoplasm; Female; Humans; Immunohistochemistry; Kidney Neoplasms; Male; Metallothionein; Middle Aged; Prognosis; Survival Rate

The antiapoptotic and mitogenic responses of metallothionein (MT) have been well documented in vitro. While MT protein overexpression, frequently encountered in a number of human primary tumors, has been shown to be correlated with disease progression, little information is available on the in vivo isoform expression of MT. In this study we have demonstrated the occurrence of MT proteins and further defined their differential expression profile in human primary renal cell carcinoma (RCC). Pooled normal human kidney RNA and paired biopsy specimens (tumor and control) obtained from 11 patients diagnosed with RCC with tumor grade ranging from 1-3 and a pathological staging of T2-T3 (N0M0) were used for the study. Samples were analyzed for the presence of MT protein using immunohistochemical (IHC) analysis and for MT isoform-specific mRNA expression by reverse transcriptase polymerase chain reaction. Metallothionein protein assumed both cytoplasmic and nuclear staining in cancer cells and was detected in eight of 11 samples (72%) with polyclonal antibodies. The immunoreactivity of MT protein, but not its cellular localization, in RCC specimens suggests a relationship between and advanced disease. While alterations in the basal level of expression of MT-1E, MT-1F and MT-1X genes remained unchanged, significant up-regulation of MT-2A and down-regulation of MT-1A and MT-1G transcripts was observed in RCC tissue specimens when compared with controls. Intriguingly, the paired RCC biopsy specimens had lower MT-1H transcripts than pooled normal human controls. We here provide the first report of the differential expression of MT isoforms in human RCC and that this data further support the role of MT-2A in tumorigenesis.

Topics: Adult; Aged; Carcinoma, Renal Cell; Disease Progression; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Kidney Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Staging; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

To investigate the frequency of apoptosis and expression of metallothionein (MT) protein and to clarify their roles in renal cell carcinoma (RCC).. Apoptosis was detected by using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labelling (TUNEL) technique in 70 formalin-fixed and paraffin-embedded RCC specimens. The expression of MT was determined immunohistochemically.. The incidence of apoptosis was significantly higher in grade 2 and 3 RCC than in grade 1. There were no significant differences amongst the other categories of RCC when grouped by tumour stage, cell type and growth pattern. The expression of MT was detected in 23 of 70 (33%) RCCs. Subcellularly. MT was localized in the cytoplasm, nucleus and cell membrane of RCC. Statistical analysis showed a close association of MT expression with the higher grades and invasive growth pattern of RCC. In addition, the incidence of apoptosis increased with increasing immunoreactivity of MT staining. There was a significant difference in the incidence of apoptosis between the diffuse staining and negative staining of MT.. There were close associations of higher grade RCC with both the incidence of apoptosis and the expression of MT, as well as a close relationship between MT expression and the invasive growth pattern of RCC. This suggests that the involvement of apoptotic cell death and MT expression should be considered an important factor in the process of the renal carcinogenesis and tumour progression.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Biomarkers; Carcinoma, Renal Cell; Female; Humans; Immunohistochemistry; Kidney Neoplasms; Male; Metallothionein; Middle Aged

To assess metallothionein (MT) expression with immunohistochemical localization in human renal cell carcinoma and to determine whether a possible relationship with the histopathologic findings, tumor grade, or pathologic tumor stage is demonstrable, because MT may have a role in carcinogenesis.. Archival pathologic specimens and medical records were reviewed for 28 patients with renal cell carcinoma. Immunohistochemical localization of MT was performed with a polyclonal-antibody-to-rat-liver MT, an anti-rabbit IgG linking antibody, and an avidin-biotin horseradish peroxidase complex. Correlation was sought between immunohistochemical data (MT staining intensity, extension, and subcellular site) and clinical data (histologic cell type, tumor grade, and pathologic stage).. The mean patient age was 61.7 years (range 42 to 86). The predominant histologic cell type was the clear cell variant. Three, sixteen, and nine tumors were pathologically staged as 1, 2, and 3, respectively. There were 1, 13, 10, and 4 tumors with grades 1, 2, 3, and 4, respectively. Among the independent variables, greater immunoreactivity was observed in Stage 2 tumors (P = 0.028). A significant inverse relationship between tumor grade and MT staining intensity was also observed (P = 0.007).. The inverse relationship in renal cell carcinoma between MT immunoreactivity and tumor grade may indicate a role for MT in tumor growth and dedifferentiation. Increased MT immunoreactivity in lower stage tumors may be related to rapid tumor growth during their growth cycle. Further study is required to elucidate the role of MT in renal cell carcinoma oncogenesis and its possible use as a clinical prognostic parameter.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Renal Cell; Female; Humans; Kidney Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Staging

Metallothionein (MT) is a low molecular-metal binding protein with multiple biological functions. Recently, MT has been implicated as a factor involved in resistance to anticancer drugs, which presumably inactivates anticancer drugs, including cisplatin, and doxorubicin. In this report, we investigated the relationship of MT expression with the clinical features in bladder cancer and renal cell carcinoma. In 35 cases of bladder cancer, 10 cases of renal cell carcinoma and 3 cases of normal mucosa of bladder, the expression of MT was immunohistologically examined by avidinebiotin-peroxidase (ABC) staining of paraffin-embedded tissue specimens with anti-MT antibody. Intense MT expression was noted in all cases of normal mucosa of bladder. MT was detected in 10 of 35 cases of bladder cancer, with the incidence of MT expression being significantly higher increases with lower pathological tumor grade. MT was detected in 8 of 10 cases of renal cell carcinoma, and all of the their normal renal tubules showed more intense staining. A number of hypotheses can be proposed from these observations. First, our observation of decreased MT expression in poorly differentiated carcinomas, which are the more proliferating tumors, this suggests correlation of MT expression with proliferative status of cancer. Second, the higher incidence of MT expression in renal cell carcinoma than in bladder cancer may suggest that it is a factor responsible for the lower efficacy of chemo-therapy in renal cell carcinoma than in bladder cancer.

Topics: Aged; Antineoplastic Agents; Carcinoma, Renal Cell; Drug Resistance; Female; Humans; Immunoenzyme Techniques; Inactivation, Metabolic; Kidney Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Urinary Bladder Neoplasms