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. 2013 Mar;5(3):366-83.
doi: 10.1002/emmm.201201161. Epub 2013 Feb 4.

A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α

Ulrike Begley  1 , Maria Soledad Sosa, Alvaro Avivar-Valderas, Ashish Patil, Lauren Endres, Yeriel Estrada, Clement T Y Chan, Dan Su, Peter C Dedon, Julio A Aguirre-Ghiso, Thomas Begley
Affiliations

A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α

Ulrike Begley et al. EMBO Mol Med. 2013 Mar.

Abstract

Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9-like (hTRM9L/KIAA1456) mRNA is down-regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re-expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence-like G0/G1-arrest and up-regulation of the RB interacting protein LIN9. Additionally, SW620 cells re-expressing hTRM9L did not respond to hypoxia via HIF1-α-dependent induction of GLUT1. Importantly, hTRM9L-negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L-expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1-α-dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L-deficient tumours.

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Figures

Figure 1
Figure 1. hTRM9L transcripts are decreased in many tumour types and the tumourgenicity of SW620 colon cancer cells can be decreased by treatment with a 5-meC demethylating agent

  1. A. Annotated gene tree of yeast Trm9 homologs. Two yTrm9 homologs are found in humans, KIAA1456(hTRM9L) and hALKBH8. In addition to the SAM-dependent Methyltransferase domain, hALKBH8 contains a RNA Recognition Motif and an AlkB domain.

  2. B. KIAA1456 (hTRM9L) expression profiling of Origene Tissue Scan qPCR array covering 18 different cancer types was used to identify significant differences in expression of hTRM9L in these cancer tissues compared to normal tissues. Significant differences in hTRM9L transcripts were evaluated and scored (Log10 p-value).

  3. C. hTRM9L expression was analysed in various colorectal cancer cell lines. RNA was isolated from SW620, SW480, HT29, HCT116 and SW1116 cell lines and hTRM9L transcript levels were quantitated (ΔΔCT method) by qPCR.

  4. D,E. HCT116 or SW620 cells were treated with 10 µM 5-aza-dC over a period of 7 days with the culture being replaced every 24 h with fresh containing 5-aza-dC. RNA was isolated and hTRM9L expression was determined by qPCR analysis.

  5. F. Mock or 5-aza-dC treated SW620 cells (5 × 105) were inoculated on CAM and grown for 7 days in vivo. Tumours were excised, minced and collagenased and the number of cells per tumour nodule was counted. Statistical significance determined by paired student's t-test.

Figure 2
Figure 2. Re-expression of hTRM9L and like methyltransferases in SW620 cells inhibits tumour growth in vivo

  1. A. 5 × 105 SW620 cells expressing either LacZ or hTRM9L were inoculated on CAM and tumours were analysed after 7 days in vivo. Tumours were excised, minced and collagenased and the number of cells per tumour nodule was counted.

  2. B,C. Experiments similar to A were performed with (B) yTrm9 and (C) mouse Alkbh8.

  3. D,E. SW620 cells expressing either LacZ or hTRM9L were inoculated in nude mice and tumours were measured every 2 days, up to 11 days. Average data for all days is plotted in (D) and day 11 data for all tumours is shown in (E). HCT116 cells expressing either LacZ or hTRM9L were inoculated in nude mice and tumours were measured every 2 days, with 12-day data shown.

  4. F. SW620 cells expressing either LacZ, hTRM9L or its single amino acid mutants hTRM9L-D91R, hTRM9L-I108N and hTRM9L-R410E were inoculated on CAM and tumours were analysed after 7 days. Tumours were excised, minced and collagenased and the number of cells per tumour nodule was counted.

Figure 3
Figure 3. hTRM9L dependent growth suppression is associated with increased cellular senescence

  1. A–E. Tumour nodules of SW620 parent, LacZ and hTRM9L expressing cells were harvested 7 days post-inoculation, fixed and prepared for (A) cleaved caspase 3, (B) LC-3 (C) phospho-Histone3 (pH3), (D) p21 and (E) p27 immunohistochemistry.

  2. F. SW620-GFP and hTRM9L expressing cells from tumour nodules were harvested after several time points as indicated and stained for acidic β-gal. Statistical significance determined by paired student's t-test.

Figure 4
Figure 4. LIN9 transcripts are up-regulated in hTRM9L expressing cells and are responsible for the growth suppression phenotype

  1. SW620 (blue circle), LacZ (green square) and hTRM9L (red triangle) tumours were harvested after several time points as indicated, minced, collagenased and quantitated as before.

  2. The intensity values measured by microarray analysis are represented in a scatter plot comparing expression patterns of SW620-LacZ (x-axis) and SW620-hTRM9L (y-axis) of 3-day-old tumours grown on the CAM. Each gene in the microarray is represented by a dot with coordinates consisting of average gene expression measured from three different replicates of RNA samples. A linear regression line is overlaid with the scatter plot and the regression equation is displayed. Outliers represent up-regulated or down-regulated genes.

  3. Real-time PCR validation of POR, LIN9, ALAS1, and hTRM9L respective expression levels. RNA was isolated from a GFP-positive population isolated from 3 day tumours grown on the CAM and amplified using MessageBOOSTER™ cDNA Synthesis Kit. Relative quantification is presented after normalization with GAPDH.

  4. Cells with a knock down of LIN9 or a scrambled control, in hTRM9L and LacZ expressing variants, were assayed for their tumour forming capacity, along with the parental cells. Statistical significance determined by paired student's t-test.

Figure 5
Figure 5. Hypoxic growth prevented in hTRM9L re-expressing cells

  1. A,B. SW620-LacZ (light grey bars) and SW620-hTRM9L (dark grey bars) expressing cells were grown under 21% oxygen (normoxic) and 1% oxygen (hypoxic) conditions and the colonies formed were quantitated. No apparent growth difference on the cell lines was detected when grown under normal oxygen conditions, whereas exposure to hypoxia revealed a growth-sensitive phenotype of hTRM9L expressing cells.

  2. C. Protein lysates prepared from 3-day-old tumours were added to MSD MULTI-SPOT 4-Spot plates coated with anti-total-HIF1-α antibody. Total HIF1-α was detected with anti-total-HIF1-α antibody labeled with MSD SULFO-TAG reagent. hTRM9L expressing tumour cells show a twofold increase in HIF1-α protein levels compared to control tumour cells.

  3. D. Immunofluorescence microscopy analysis of HIF1-α in hTRM9L deficient and proficient cells under normoxic and hypoxic conditions. Images were not fixed in exposure time, thus, they do not reflect the quantitative protein abundance difference shown in (C).

  4. E. Levels of downstream markers for HIF1-α target activation, GLUT1, were analysed in both cells types.

Figure 6
Figure 6. hTRM9L affects tRNA modification and prevents aminoglycoside antibiotic induced death

  1. Complementation analysis of trm9Δ yeast cells with hTRM9L, hALKBH8 and yTrm9.

  2. Hierarchical clustering of tRNA modification data, with significance calculated relative to trm9Δ cells. tRNA modification increases were assigned a positive p-value, while decreases were assigned a negative p-value.

  3. SW620-lacZ and SW620-hTRM9L cells were left untreated or exposed to 1.5 mg/ml paromomycin for 24 h. For data reported in (B) and (C), small RNA was isolated and validated to be of high integrity by Bioanalyser analysis. RNA was enzymatically digested to nucleosides and the identity and levels, as determined by total ion count (TIC), of specific modified nucleosides was determined by LS/MS-MS analysis.

  4. SW480 and SW620 cells were treated with paromomycin and the percent viability for each was determined by trypan blue staining 24 h post exposure.

  5. SW620-LacZ and SW620-hTRM9L cells were exposed to paromomycin and assays were performed as described above.

Figure 7
Figure 7. Proposed mechanism of growth suppression in hTRM9L proficient cells
Direct or indirect regulation of LIN9 and the hypoxic response lead to decreased tumour growth in hTRM9L positive cells.
See this image and copyright information in PMC

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