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The biological process of lysine-tRNA charging is therapeutically targetable in liver cancer - PubMed

The biological process of lysine-tRNA charging is therapeutically targetable in liver cancer

Ruyi Zhang et al. Liver Int. 2021 Jan.

Abstract

Background & aims: Mature transfer RNAs (tRNA) charged with amino acids decode mRNA to synthesize proteins. Dysregulation of translational machineries has a fundamental impact on cancer biology. This study aims to map the tRNAome landscape in liver cancer patients and to explore potential therapeutic targets at the interface of charging amino acid with tRNA.

Methods: Resected tumour and paired tumour-free (TFL) tissues from hepatocellular carcinoma (HCC) patients (n = 69), and healthy liver tissues from organ transplant donors (n = 21), HCC cell lines, and cholangiocarcinoma (CC) patient-derived tumour organoids were used.

Results: The expression levels of different mature tRNAs were highly correlated and closely clustered within individual tissues, suggesting that different members of the tRNAome function cooperatively in protein translation. Interestingly, high expression of tRNA-Lys-CUU in HCC tumours was associated with more tumour recurrence (HR 1.1; P = .022) and worse patient survival (HR 1.1; P = .0037). The expression of Lysyl-tRNA Synthetase (KARS), the enzyme catalysing the charge of lysine to tRNA-Lys-CUU, was significantly upregulated in HCC tumour tissues compared to tumour-free liver tissues. In HCC cell lines, lysine deprivation, KARS knockdown or treatment with the KARS inhibitor cladosporin effectively inhibited overall cell growth, single cell-based colony formation and cell migration. This was mechanistically mediated by cell cycling arrest and induction of apoptosis. Finally, these inhibitory effects were confirmed in 3D cultured patient-derived CC organoids.

Conclusions: The biological process of charging tRNA-Lys-CUU with lysine sustains liver cancer cell growth and migration, and is clinically relevant in HCC patients. This process can be therapeutically targeted and represents an unexplored territory for developing novel treatment strategies against liver cancer.

Keywords: Lysyl-tRNA Synthetase; cladosporin; liver cancer; lysine; tRNA-Lys-CUU; tRNAome.

© 2020 The Authors. Liver International published by John Wiley & Sons Ltd.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Profiling tRNAome in HCC patients identified tRNA‐Lys‐CUU having potential clinical significance. (A) Heatmap showing the mean expression (z‐score normalized per tRNA species, represented in colour, as indicated in the legend) of 57 tRNA in tumour and TFL tissues respectively. Both rows (tRNA) and columns (patient samples) of tRNAs expressed in tumours (left panel) are hierarchically clustered using correlation distance and complete linkage. Rows and columns of tRNAs expressed in TFL (right panel) were ordered according to the rows and columns in the tumour heatmap. (B and C) Kaplan‐Meier analysis and Cox regression analysis of tumour recurrence (B) and HCC‐specific survival (C) in relation to tRNA‐Lys‐CUU expression. The panels below the survival graphs show the results of univariate Cox regression analysis. HR, Hazard Ratio; AAC, amino acid class; HCC, hepatocellular carcinoma; TFL, tumour free liver; CI, confidence interval

Figure 2
Figure 2

KARS1 expression and clinical relevance in HCC patients. (A) mRNA expression of KARS1 in HCC tumours compared to paired TFL tissues (n = 59). (B and C) Kaplan‐Meier analysis of tumour recurrence (B) and HCC‐specific survival (C) in relation to KARS1 expression in tumours. The panels below the survival graphs show the results of univariate Cox regression analysis. (D‐F) Correlation analysis between the expression levels of tRNA‐Lys‐CUU and KARS1 in healthy organ donor liver tissues (D, n = 21), TFL (E) and paired HCC tissues (F, n = 59) respectively. **P < .01, by the Wilcoxon matched pairs test. KARS1, Lysyl‐tRNA Synthetase; HCC, hepatocellular carcinoma; TFL, tumour free liver; HR, Hazard Ratio; CI, confidence interval

Figure 3
Figure 3

The effects of KARS knockdown on HCC cell lines. (A) Relative expression of KARS1 mRNA in HCC cell lines compared to SNU398 (n = 4‐6). (B) KARS protein expression in HCC cell lines. (C) Protein expression levels of KARS and cleaved‐Caspase 3 in Huh7 and SNU398 HCC cells following shRNA mediated knockdown of KARS. The intensity was quantified relative to β‐actin (n = 4‐8). (D) KARS1 mRNA expression following shRNA mediated knockdown quantified by qRT‐PCR (n = 4‐10). (E) Effects of KARS knockdown on cell growth measured by MTT assay following 1, 3 and 6 days of culture (n = 11, 22). (F) KARS knockdown affects the number of single cell formed colonies as assayed 2 weeks following seeding (n = 5). (G) The effect of KARS knockdown on cell cycling was measured by Propidium Iodide staining, and the fraction of cells in G0/G1 was quantified (n = 4‐6). Quantification of C, D and F data of knockdown groups were relative to the shCTR group. Data are presented as mean ± SEM. *P < .05; **P < .01; ***P < .001, by the Mann‐Whitney test. HLO, healthy liver organoids; HCC, hepatocellular carcinoma; FC, fold change; KARS1, Lysyl‐tRNA Synthetase

Figure 4
Figure 4

The effects of lysine on HCC cell lines. (A) The effects of lysine on cell growth measured by MTT assay following 1, 3 and 6 days of culture (n = 20). (B) Lysine affects the number of single cell‐derived colonies as assayed 2 weeks following seeding (n = 6). (C) The effects of lysine on cell cycling was measured by Propidium Iodide staining, and the fraction of cells in G0/G1 was quantified (n = 6‐9). (D) Protein expression levels of cleaved‐Caspase 3 in Huh7 and SNU398 HCC cells. The intensity was quantified relative to β‐actin (n = 5). Huh7 and SNU398 cells were cultured with or without lysine for 3 days. (E) Representative images of migrating cells with or without lysine and (F) quantification of the number of migrating cells (n = 10‐20). Quantification of A, B, D and F data without lysine groups were relative to with lysine group. Data are presented as mean ± SEM. *P < .05; **P < .01; ***P < .001, by the Mann‐Whitney test. HCC, hepatocellular carcinoma; FC, fold change

Figure 5
Figure 5

The effects of cladosporin treatment on HCC cell lines. (A) Huh7 and SNU398 cells were treated with different concentrations of cladosporin for 3 days. Cell viability was measured by MTT assay (n = 12). (B) Cladosporin affects the number of single cell‐derived colonies as assayed 2 weeks following seeding (n = 6‐8). (C) Protein expression levels of cleaved‐Caspase 3 in Huh7 and SNU398 cells, and the intensity was quantified relative to β‐actin (n = 5). Huh7 and SNU398 were treated with cladosporin for 3 days. (D) Representative images of migrating cells with cladosporin and (E) quantification of the number of migrating cells (n = 20‐25). Quantification of all data were relative to the negative control group. Data are presented as mean ± SEM. *P < .05; **P < .01; ***P < .001, by the Mann‐Whitney test. HCC, hepatocellular carcinoma; FC, fold change

Figure 6
Figure 6

Lysine deprivation or treatment with cladosporin affects the growth of patient‐devied CC organoids. (A) Relative expression of KARS1 mRNA in patient CC organoids and human healthy liver organoids (HLO) (n = 5‐11). Effects of (B) lysine deprivation or (C) cladosporin treatment on cell viability were measured by Alamar blue assay after culturing 3 or 7 days (n = 4‐12). Organoids were treated with (D) lysine deprivation or (E) cladosporin (100 µM) for 7 or 14 days. The results depicts the mean viability of CC organoids (CCO‐1, CCO‐2, CCO‐3) compared with the mean viability of healthy liver organoids (HLO‐1, HLO‐2, HLO‐3) (n = 18‐24). (F) Lysine deprivation or (G) cladosporin treatment affects organoid initiation as assayed 2 weeks following seeding. The number (n = 4‐10) and size (n = 18‐45) of organoids after culturing for 14 days were calculated. Quantification of the data target groups were relative to the negative control group. Data are presented as mean ± SEM and dots represent individual organoid cultures. *P < .05, **P < .01, ***P < .001, by the Mann‐Whitney test. CCO, cholangiocarcinoma organoid; HLO, human liver healthy organoid; FC, fold change

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