Elp3 links tRNA modification to IRES-dependent translation of LEF1 to sustain metastasis in breast cancer

Abstract

Quantitative and qualitative changes in mRNA translation occur in tumor cells and support cancer progression and metastasis. Posttranscriptional modifications of transfer RNAs (tRNAs) at the wobble uridine 34 (U34) base are highly conserved and contribute to translation fidelity. Here, we show that ELP3 and CTU1/2, partner enzymes in U34 mcm⁵s²-tRNA modification, are up-regulated in human breast cancers and sustain metastasis. Elp3 genetic ablation strongly impaired invasion and metastasis formation in the PyMT model of invasive breast cancer. Mechanistically, ELP3 and CTU1/2 support cellular invasion through the translation of the oncoprotein DEK. As a result, DEK promotes the IRES-dependent translation of the proinvasive transcription factor LEF1. Consistently, a DEK mutant, whose codon composition is independent of U34 mcm⁵s²-tRNA modification, escapes the ELP3- and CTU1-dependent regulation and restores the IRES-dependent LEF1 expression. Our results demonstrate that the key role of U34 tRNA modification is to support specific translation during breast cancer progression and highlight a functional link between tRNA modification- and IRES-dependent translation during tumor cell invasion and metastasis.

Figure 1.

U34 tRNA-modifying enzymes are up-regulated in human breast cancer. (A–C) Tissue microarray analysis of ELP3 (A), CTU2 (B), and CTU1 (C) protein expression in normal breast tissue (n = 23) and in samples of noninvasive or invasive human breast cancer (n = 35 and n = 7, respectively). Representative images are shown. The mean Elp3-specific signal has been quantified and plotted (mean values ± SD; Student’s t test; *, P < 0.05; ***, P < 0.001). (D) IHC staining for Elp3, Ctu1, and Ctu2 expression in normal mammary glands of 7-wk-old mice. (E) IHC staining for Elp3, Ctu1, and Ctu2 expression in mammary glands from 7-wk-old PyMT mice. (F) Western blot showing Elp3, Elp1, Ctu1/2, and Alkbh8 expression in control NMuMG cells or after stable expression the PyMT oncogene. pAkt is used as a control to prove that PyMT expression was functional. Total Akt and Hsp90 were used as loading controls. (G) RT-qPCR analysis of PyMT mRNA in control or PyMT-expressing NMUMG cells. Detection of the GAPDH mRNA was used as normalization. The expression of PyMT in control cells was set to 1, and values obtained from PyMT-NMUMG cells expressed relative to it (**, P ≤ 0.01, Student's t test). (H) Northern blot analysis assessing t:E(UUC) tRNA thiolation in control and PyMT-expressing NMuMG cells. (I) Quantification of t:E(UUC) tRNA thiolation, calculated as the ration of thiolated over nonthiolated t:E(UUC), in control and PyMT-expressing NMUMG cells. Values represent mean ± SD of three independent experiments (*, P ≤ 0.05, Student’s t test).

Figure 2.

Breast cancer metastasis relies on U34 tRNA modifications. (A) IHC staining for Elp3 expression in normal mammary glands from 7-wk-old Elp3^CTR or Elp3^ΔMEC mice. (B) RT-qPCR analysis of Elp3 mRNA levels in 7-wk Elp3^CTR and Elp3^ΔMEC mammary glands. Values represent mean ± SD of five different tumors performed in triplicate (n = 5; *, P ≤ 0.05, Student’s t test). (C) Representative whole mounted normal mammary glands from 8-wk Elp3^CTR and Elp3^ΔMEC mice. (D) Quantification of terminal end buds (TEB) from whole-mounted normal mammary glands from 8-wk Elp3^CTR and Elp3^ΔMEC mice (n = 5 and n = 7, respectively; ns, nonsignificant). (E) RT-qPCR analysis of Elp3 mRNA levels in 7-wk Elp3^CTRPyMT and Elp3^ΔMECPyMT tumors. Values represent mean ± SD of five different tumors performed in triplicate (**, P ≤ 0.01, Student’s t test). (F) Kaplan-Meier curve showing tumor appearance in Elp3^CTRPyMT and Elp3^ΔMECPyMT mice (n = 12 per condition; ***, P ≤ 0.001). (G) Quantification of total tumor burden in 14 wk Elp3^CTRPyMT (n = 8) and Elp3^ΔMECPyMT mice (n = 8) or in 16 wk Elp3^ΔMECPyMT (n = 5). Tumor weight values are expressed as a percentage of the total mouse weight (*, P ≤ 0.05, Student’s t test). (H) Quantification of tumors surface in mammary gland of 5, 7, 9, and 11 wk Elp3^CTRPyMT (n = 3 per time point) and Elp3^ΔMECPyMT (n = 3 per time point) mice based on H&E staining (***, P ≤ 0.001, Student’s t test). (I) Quantification of Ki67 staining in tumors of 7 and 14 wk Elp3^CTRPyMT and Elp3^ΔMECPyMT mice. The percentage of Ki67-positive cells in tumors is expressed (mean values ± SD; five mice per condition). (J) Anti-Elp3, Ctu1, and Ctu2 IHC analyses in metastazed lungs from 14-wk-old Elp3^CTRPyMT mice. A representative picture is shown. (K) Representative lungs and corresponding H&E staining of 14 wk Elp3^CTRPyMT and Elp3^ΔMECPyMT mice and of 16 wk Elp3^ΔMECPyMT mice. (L) Quantification of the number of Elp3^CTRPyMT and Elp3^ΔMECPyMT mice with detectable lung metastases. (M and N) Quantification of the number of lung metastases (M) and surface metastases (N) in 14 wk Elp3^CTRPyMT (n = 9) and Elp3^ΔMECPyMT (n = 11) mice and in 16 wk Elp3^ΔMECPyMT mice (n = 5; *, P ≤ 0.05; **, P ≤ 0.01, Student’s t test). (O) Quantification of the number of mice with β-casein–positive lung in 5, 7, 9, and 11 wk Elp3^CTRPyMT (n = 3 per time point) and Elp3^ΔMECPyMT (n = 3 per time point). β-casein mRNA was detected by RT-qPCR using the GAPDH mRNA for normalization purposes.

Figure 3.

U34 tRNA modifications regulate breast cancer cell invasion. (A) Representative pictures of ex vivo 3D collagen I culture of organoids from similarly sized 9-wk-old Elp3^CTRPyMT and Elp3^ΔMECPyMT primary tumors. (B and C) Quantification of the number of leader cells per organoid (B) and the mean length of leader cells (C). Data are mean ± SD (n = 3 biological replicates per group; *, P ≤ 0.05; ***, P ≤ 0.001, Student’s t test). (D) MCF7 cells were cultured in nonadherent conditions for several weeks and passaged 4 times (M1 to M4). Resulting cells were then cultured in adherent conditions (MCF7-M). Representative pictures are shown. (E) Migration of control or ELP3-depleted MCF7-M cells toward a serum gradient was measured using Transwell assays. The results are expressed as number of migrating cells per field. Values represent mean ± SD of three independent experiments performed in triplicates (***, P ≤ 0.001). (F) Western blot showing ELP3, ELP1, CTU1/2, ALKBH8, Vimentin, and E-Cadherin expression in MCF7 and MCF7-M cells. HSP90 was detected for normalization purpose. (G) Migration of control and ELP3-depleted MCF7-M cells toward a serum gradient was measured using Transwell assays. The results are expressed as in E (**, P ≤ 0.01, Student’s t test). (H and I) Migration of control, ELP3- (H), or CTU1-depleted (I) MDA-MB-231 cells toward a serum gradient was measured using Transwell assays. The results are expressed as in E (*, P ≤ 0.05; **, P ≤ 0.01, Student’s t test). (J) Northern blot analysis assessing t:E(UUC) tRNA thiolation in control or ELP3-depleted MDA-MB-231 cells. (K) Quantification of t:E(UUC) tRNA thiolation, calculated as the ratio of thiolated over nonthiolated t:E(UUC), in control and ELP3-depleted cells. Values represent mean ± SD of three independent experiments (**, P ≤ 0.01; ***, P ≤ 0.001, Student’s t test).

Figure 4.

Elp3-deficient PyMT tumors lack a Lef1-dependent gene signature. (A) Scatter plot of RNA-seq data obtained from in Elp3^CTRPyMT and Elp3^ΔMECPyMT tumor cells. (B) RT-qPCR analysis was used to assess the expression of the indicated gens in Elp3^CTRPyMT and Elp3^ΔMECPyMT tumor cells. Values represent mean ± SD of signals form three different mice, performed in triplicate (***, P ≤ 0.001, Student’s t test). (C) GSEA analyses of published metastasis- and invasion-related datasets for genes down-regulated in Elp3^ΔMECPyMT tumors. NES, normalized enrichment score with adjusted p-value for each enrichment plots. (D) GSEA analysis using Oncogenic Pathway database. NES and adjusted p-values are added for each enrichment dataset. (E) Enrichment plot of the Lef1 dataset (with NES and p-value). Heatmap of the top 20 genes down-regulated in Elp3^ΔMECPyMT from the Lef1 dataset. (F) Western blot analysis of Elp3, Lef1, and Ctu1 in similarly sized Elp3^CTRPyMT and Elp3^ΔMECPyMT tumors (14 wk). (G) RT-qPCR analysis of Lef1 mRNA and its target genes (Pik3r5, Ephb2, PdgfrA, and Vcam). Values represent mean ± SD of signals from five different 14 wk tumors, performed in triplicate (*, P ≤ 0.05, Student’s t test). (H) LEF1, ELP3, and CTU1 protein levels in control (CTR) or LEF1-depleted (LEF1#1 and LEF1#2) MDA-MB231 cells were assessed by Western blot analysis. (I) Migration of control (shCTR) or LEF1-depleted (shLEF1#1 and shLEF#2) MDA-MB-231 cells toward a serum gradient was measured using Transwell assays. The results are expressed as number of migrating cells per field. Values represent mean ± SD of three independent experiments performed in triplicates (*, P ≤ 0.05; **, P ≤ 0.01, Student’s t test).

Figure 5.

U34 tRNA modifications regulate LEF1 IRES translation to promote breast cancer cell migration. (A) Western blot analysis of indicated proteins in Py2T cells (extracted from PyMT tumors) and Py2T-LT (idem and long-time stimulated with TGFβ). HSP90 is used as loading control. (B) Western blot analysis of LEF1 and ELP3 in MCF7 and MCF7-M cells. HSP90 is used as loading control. (C and D) ELP3 and LEF-1 protein (C) or mRNA (D) levels in control (CTR) or ELP3-depleted (ELP3#1 and ELP3#2) MCF7-M, Py2T-LT, and MDA-MB-231 cells as indicated. Values represent mean ± SD of three experiments performed in triplicate (*, P ≤ 0.05; ***, P ≤ 0.001; ns, nonsignificant, Student’s t test). (E) Representative polysome profiles of control (shCTR) or ELP3-depleted (shELP3#1 and ELP3#2) MCF7-M cells. (F) As in E, but translation efficiency, calculated as the ratio of polysomal (P) compared with subpolysomal fractions (S), is shown in the graph. Data are mean ± SD (n = 3 per group; *, P ≤ 0.05, Student’s t test). (G) MCF7-M cells were pulsed for 30 min with ³⁵S-labeled methionine/cysteine. Incorporation of ³⁵S into protein was quantified by scintillation counting and normalized to total amount of proteins. Data are mean ± SD (n = 3 biological replicates per group; *, P ≤ 0.05, Student’s t test). (H) Distribution of LEF-1–specific mRNA in polysome fractions quantified by RT-qPCR. Data are mean ± SD of three independent experiments performed in triplicates. (I) Quantification of LEF-1–specific translation efficiency by calculation of the ratio of polysomal (P) compared with subpolysomal (S) fractions. Data are mean ± SD (n = 3 per group; *, P ≤ 0.05; ***, P ≤ 0.001, Student’s t test). (J) Western blot showing FLAG and ELP3 expression in control (shCTR) or ELP3-depleted (shELP3#1 and shELP3#2) MDA-MB-231 cells stably expressing empty vector (EV), IRES-LEF1 cDNA (IRES-LEF1-FLAG), or LEF1 cDNA (LEF1-FLAG). Numbers represent ratios between the respective FLAG and HSP90 signals. (K) Migration of control or ELP3-depleted MDA-MB-231 cells stably expressing IRES-LEF1 cDNA (IRES-LEF1-FLAG) or LEF1 cDNA (LEF1-FLAG) toward a serum gradient was measured using Transwell assays. Representative pictures are shown. Quantification of migrating cells is also shown in the graph (right). Results are expressed as number of migrating cells per field. Values represent mean ± SD of three representative experiments performed in triplicates (*, P ≤ 0.05; ***, P ≤ 0.001, Student’s t test).

Figure 6.

The ITAF DEK is a direct target of U34 tRNA modifications. (A) Table of reported LEF1 ITAFs (Tsai et al., 2011). Proteins were ordered according to the combined frequency of AAA, GAA, and CAA codons in their ORF. (B) Western blot showing indicated proteins expression in control (shCTR) or ELP3-depleted (shELP3#1 and shELP3#2) MDA-MB-231 cells. HSP90 is used as loading control. (C) RT-qPCR assessing DEK mRNA levels in control or ELP3-depleted MDA-MB-231 cells. Values represent mean ± SD of three different experiments performed in triplicate. (D and E) Expression of LEF1 and DEK was assessed at protein levels by Western blot (D and F) and at mRNA levels by RT-qPCR (E and G) in control or ELP3-depleted MCF7-M cells. (F–I) DEK and LEF1 protein (F and H) and mRNA (G and I) expression were assessed in control or CTU-1–depleted MDA-MB-231 (F and G) and MCF7-M cells (H and I; ***, P < 0.001, Student’s t test). (J) Western blot showing DEK protein expression in extracts from Elp3^CTRPyMT and Elp3^ΔMECPyMT primary tumors of similar size. HSP90 is used as loading control. (K) IHC staining for Dek expression in tumorigenic mammary glands from 7-wk-old Elp3^CTRPyMT or Elp3^ΔMECPyMT mice. Images were generated from five different tumors/mouse, using three mice per condition. Representative images are shown. (L) The mean Dek-specific signal shown in M was quantified and plotted (mean values ± SD; Student’s t test; ***, P < 0.001). (M) Positions of AAA, CAA, and GAA codons were represented in the DEK ORF sequence. (N) DEK (FLAG), LEF1, ELP3, and CTU1 protein levels were assessed in control (shCTR), ELP3 (shELP3)-, or CTU1 (shCTU1)-depleted MDA-MB-231 stably expressing DEK-wild-type sequence (DEK-WT-FLAG) or a DEF mutant in which AAA, CAA, and GAA codons have been replaced by their synonymous codons (AGA, CAG, GAG, respectively; DEK-δAAA/GAA/CAA-FLAG).

Figure 7.

The ITAF DEK is required for IRES-LEF1 protein synthesis. (A) Kaplan-Meier curves showing distant metastasis-free survival according to DEK mRNA expression using GOBO (Gene expression-based Outcome for Breast cancer Online; Ringnér et al., 2011). Black, low expression; gray, medium expression; red, high expression (n represents the number of patients included in the analysis). (B) Box plot graph showing DEK mRNA expression in breast cancers of different grades according to GOBO. (C) Western blot showing expression of indicated proteins in MDA-MB-231 cells transfected with esiRNAs control or targeting DEK. (D) Same as C, but detecting LEF1 and DEK mRNA levels by RT-qPCR. (E) Western blot showing expression of indicated proteins in MDA-MB-231 cells stably expressing shRNA control or specific to DEK (DEK#1 and DEK#2). (F–H) Luciferase reporter assay using a bicistronic Renilla luciferase-IRES-LEF1-firefly luciferase construct (i.e., pRSTF-LEF1) in control (CTR) and ELP3- (G) or DEK-depleted (H) MDA-MB-231 cells (ELP3#1, ELP3#2 and DEK#1, and DEK#2). Values represent mean ± SD of three independent experiments performed in triplicate. (I and J) RT-qPCR showing the ratio of mRNA levels of the firefly amplicon (Fire1), selected close to the AUG initiation codon, on two renilla amplicons (Ren1 and Ren2) in control (CTR) and ELP3- (G) or DEK-depleted (H) MDA-MB-231 cells (ELP3#1, ELP3#2 and DEK#1, and DEK#2). Values represent mean ± SD of three independent experiments performed in triplicate. (K and L) Luciferase reporter assay using pRTSF-LEF1 construct or the corresponding promoterless construct pRSTF-LEF1^ΔSV40 in MDA-MB-231 cells. Data are expressed as the relative light unit of firefly, normalized to β-gal levels, as a control of transfection efficiency. Values represent mean ± SD of three different experiments performed in triplicate. (M) RT-qPCR showing mRNA levels of the firefly mRNA normalized to GAPDH levels in MDA-MB-231 cells, transfected with pRSTF-LEF1 or pRSTF-LEF1^ΔSV40. Values represent mean ± SD of three different experiments performed in triplicate. (N) Migration of control (shCTR) or DEK-depleted (DEK#1 and DEK#2) MDA-MB-231 cells toward a serum gradient was measured using Transwell assays. The results are expressed as number of migrating cells per field. Values represent mean ± SD of three independent experiments performed in triplicates.

Figure 8.

Expression of ELP3, LEF1, and DEK correlates in human breast cancer patients. (A) Western blot analysis of ELP3, LEF1, and DEK protein levels in the indicated human breast cancer cell lines. HSP90 is detected for normalization purpose. (B) Expression values (relative OD) of all human breast cancer samples were plotted and paired correlations were calculated. The table summarizes the correlation values (R). (C–E) ELP3, LEF1, and DEK protein levels were assessed in ER⁺ (C), HER2⁺ (D), and Triple-negative breast cancer (TNBC; E) human breast cancer samples. α-Tubulin and HSP90 were used as loading controls. (F) Expression values (relative OD) of all human breast cancer samples were plotted and paired correlations were calculated. The table summarizes the correlation values (R). (G–I) Kaplan-Meier curves for distant metastasis-free survival to all sites according to concomitant ELP3, DEK, and LEF1 mRNA expression, using GEO accession nos. GSE26971 (G), GSE11121 (H), GSE2990 (I) datasets of breast cancer patients. Patients were stratified into low and high expression based on autoselect best cutoff. Green, low expression; red, high expression. P-values were calculated with log-rank (Mantel–Cox) test.

Figure 9.

Model for the implication of the U34 tRNA modifications in breast cancer metastasis. The U34 tRNA-modifying enzymes ELP3 and CTU1/2 are induced in models of invasive breast cancer and are critical for the translation of DEK, which in turn regulates the IRES-dependent translation of the oncogenic LEF-1 mRNA to promote breast cancer cells motility and metastasis. This mechanism is essential for the establishment of a LEF-1–dependent prometastatic genes signature in breast tumors.