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The endogenous retrovirus-derived long noncoding RNA TROJAN promotes triple-negative breast cancer progression via ZMYND8 degradation

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Science Advances  06 Mar 2019:
Vol. 5, no. 3, eaat9820
DOI: 10.1126/sciadv.aat9820
  • Fig. 1 HERV transcriptome profile in human breast cancer.

    (A) Schematic diagram depicting the screening of TNBC-related HERVs. RNA-seq detected HERV expression across eight paired TNBC tissues and adjacent normal tissues for HERVs derived from RepeatMasker. FPKM, fragments per kilobase of transcript per million mapped reads. (B) The top 5 TNBC-related HERVs identified by RNA-seq. (C) The expression of LTR70 [chromosome (chr) 19: 20331250-20332477] in eight paired TNBC tissues and adjacent normal tissues. P value was determined using two-tailed paired Student’s t test. (D) Polymerase chain reaction (PCR) products generated in the 3′ (left) and 5′ (right) RACE assay covering the 5′ and 3′ ends of the TROJAN transcript. (E) The quantitative PCR (qPCR) analysis of the relative TROJAN transcription levels in TNBC tissues (n = 53) versus the adjacent normal breast tissues (n = 53) in FUSCC cohort 1. P value was determined using two-tailed paired Student’s t test. (F) Kaplan-Meier analysis of the relapse-free survival of 153 patients with TNBC in FUSCC cohort 1. A log-rank test was used to determine the statistical significance between the low TROJAN expression group (n = 51) and the high TROJAN expression group (n = 102). (G) RNA ISH of TROJAN in breast cancer tissues with different subtypes (n = 50 each) (FUSCC cohort 2). Scale bars, 50 μm. The data are presented as the median with interquartile range; two-tailed unpaired Student’s t test. **P < 0.01 and ***P < 0.001. (H) Kaplan-Meier analysis of the relapse-free survival of 50 patients with TNBC in FUSCC cohort 2. The log-rank test was used to determine statistical significance between the low TROJAN expression group (n = 31) and the high TROJAN expression group (n = 19). (I) The constituent ratio of LTR70. The assay was performed by RNA-seq. (J) The qPCR analysis of the expression of TROJAN and two other LTR70s in multiple cell lines. The data are presented as the mean ± SD; n = 3 independent experiments. See also figs. S1 and S2.

  • Fig. 2 The oncogenic role of TROJAN in breast cancer.

    (A) In vitro growth curves of the breast cancer cell line MDA-MB-231 LM2 expressing control (Ctrl) or TROJAN shRNAs. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. (B) In vitro growth curves of the breast cancer cell line BT549 expressing a control empty vector (Vec) or TROJAN. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. OD450, optical density at 450 nm. (C) In vivo growth of MDA-MB-231 LM2 (39 days; n = 5) expressing control or TROJAN shRNAs. Tumor volume quantification (left) and representative tumor images (right) are shown. The data are presented as the mean ± SD; two-tailed unpaired Student’s t test. (D and E) In vitro transwell migration assay of MDA-MB-231 LM2 (D) and BT549 (E) cells expressing control or TROJAN shRNAs. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. Scale bars, 200 μm. (F) In vitro transwell invasion assay of MDA-MB-231 LM2 cells expressing control or TROJAN shRNAs. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. Scale bars, 200 μm. (G) In vivo intravenous xenograft mouse model of the MDA-MB-231 LM2 (42 days; n = 5) cells expressing control or TROJAN shRNAs. The relative bioluminescence intensities (BLIs) indicate lung metastasis. The data are presented as the mean ± SD; two-tailed unpaired Student’s t test. (H) BLI of bone metastasis of SCP2 cells expressing control or TROJAN shRNA. The data are presented as the mean ± SE; Wilcoxon signed-rank test. **P < 0.01 and ***P < 0.001. See also figs. S3 and S4.

  • Fig. 3 Potential therapeutic role of TROJAN in breast cancer progression.

    (A) Quantitative reverse transcription PCR (qRT-PCR) analysis of the relative TROJAN transcription in the MDA-MB-231 LM2 cells transfected with ASOs targeting TROJAN or control. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. (B) In vitro growth assay of MDA-MB-231 LM2 cells transfected with ASOs targeting TROJAN or control. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. h, hours. (C) In vitro free uptake assay in the MDA-MB-231 LM2 cells. Six different concentrations of ASO were assessed (0, 0.5, 1, 2.5, 5, and 10 μM). The 0 μM group was set as the control. qRT-PCR detection of relative TROJAN expression. The data are presented as the mean ± SD; n = 3 independent experiments. (D) Illustration of the TROJAN-targeted ASO treatment timeline. Briefly, we injected MDA-MB-231 LM2 cells into the tail veins of female BALB/c nude mice to construct a lung metastasis model. Approximately 28 days after injection, we randomly assigned eight mice with similar lung BLI signals to two groups, the PBS and ASO treatment groups. Both groups received treatment (PBS or ASO) twice per week immediately after randomization, and BLI imaging was performed once per week. The arrows indicate the different events (green, treatment; red, bioluminescence imaging). (E and F) Lung metastasis quantification (E), gross anatomy, hematoxylin and eosin (H&E) lung staining, and number of metastatic nodules in the lungs (F) are shown. The data are presented as the mean ± SD; two-tailed unpaired Student’s t test. Scale bars, 200 μm. (G) The TROJAN-targeted ASO treatment timeline and the tumor volumes of the three treatment groups (PBS, ASO-1, and ASO-4) are shown. Briefly, we injected MDA-MB-231 LM2 cells into the mammary fat pads of female NOD/SCID mice to construct an orthotopic xenograft model. Approximately 21 days after injection, we randomly assigned nine mice with similar tumor volumes to three treatment groups: PBS, ASO-1, and ASO-4 groups. All groups received treatment (PBS, ASO-1, or ASO-4) twice per week immediately after randomization. The green arrows indicate the treatments. The data are presented as the mean ± SD; two-tailed unpaired Student’s t test. (H) Representative tumor images are shown. *P < 0.05, **P < 0.01, and ***P < 0.001. NS, not significant. See also fig. S5.

  • Fig. 4 TROJAN associates with the ZMYND8 protein.

    (A) RIP assay and the subsequent qRT-PCR assay and semi–RT-PCR (inset). Relative quantification of TROJAN and MALAT1 expression in MDA-MB-231 LM2 RNA-protein complexes immunoprecipitated with IgG or ZMYND8 antibodies. MALAT1 was used as a negative control. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. (B) Confocal RNA fluorescence ISH and immunofluorescence images showing the colocalization of TROJAN and ZMYND8. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (C) Schematic representation of Flag-tagged full-length human ZMYND8 and its deletion mutants. The ΔN, ΔInter, and ΔC constructs are depicted. The anti-Flag Western blot image showing ZMYND8 full-length or deletion mutant expression in human embryonic kidney (HEK) 293T cells. (D) RIP assay and the subsequent qRT-PCR assay performed in HEK293T cells ectopically expressing full-length Flag-tagged ZMYND8 and its deletion mutants. Relative quantification of TROJAN expression in RNA-protein complexes immunoprecipitated with IgG or Flag antibodies. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. (E) TROJAN motif and schematic representation of full-length TROJAN and its deletion mutants. (F) RIP assay and the subsequent qRT-PCR assay. HEK293T cells were cotransfected with full-length TROJAN (FL)/mutants (Δ1 to Δ4)/control vector as well as Flag-ZMYND8/Flag-vector (Flag-Vec). After 48 hours, the cell lysates were immunoprecipitated with beads coated with a Flag antibody. Two primers (#1 for FL, Δ1, and Δ4 and #2 for Δ2 and Δ3) were used in the qRT-PCR assay. Fold enrichment was adjusted by each input and Flag-Vec. The data are presented as the mean ± SD; n = 3 independent experiments; two-tailed unpaired Student’s t test. *P < 0.05 and ***P < 0.001. See also fig. S6.

  • Fig. 5 TROJAN regulates ZMYND8 protein stability via ubiquitination.

    (A) Western blot images showing ZMYND8 expression in MDA-MB-231 LM2 cells expressing control or TROJAN shRNAs. (B) Western blot images showing ZMYND8 expression in MDA-MB-231 LM2 cells expressing control or TROJAN shRNA treated with dimethyl sulfoxide (DMSO) (control) or MG132. (C) Western blot images showing ZMYND8 expression in BT549 cells expressing control or TROJAN shRNAs. (D) Western blot images of ZMYND8 expression in BT549 cells expressing control or TROJAN shRNAs treated with DMSO or MG132. (E) Western blot images showing ZMYND8 expression in MDA-MB-231 LM2 cells overexpressing control or TROJAN treated with DMSO or MG132. (F) Western blot images of ZMYND8 expression in MDA-MB-231 LM2 cells expressing control or TROJAN shRNAs treated with DMSO or CHX. (G) Western blot images showing ZMYND8-associated ubiquitination (Ubi) in control and TROJAN–down-regulated HEK293T cells ectopically expressing full-length Flag-tagged ZMYND8 treated with MG132. HA, hemagglutinin. (H) In vivo intravenous xenograft mouse model in MDA-MB-231 LM2 cells expressing TROJAN and/or ZMYND8 shRNAs (n = 6 for each group). The data are presented as the mean ± SD; two-tailed unpaired Student’s t test. ***P < 0.001. (I) Western blot images showing the interaction of ZNF592 with ZMYND8 under TROJAN knockdown. See also figs. S7 and S8.

  • Fig. 6 Correlation among TROJAN, ZMYND8, and tumor progression in clinical samples.

    (A) IHC detection of ZMYND8 in FUSCC cohorts 1 and 2. Scale bars, 100 μm. (B and C) Correlation between TROJAN and ZMYND8 expression in FUSCC cohorts 1 (B) and 2 (C), as determined by Pearson correlation analysis. (D and E) Kaplan-Meier analysis of the relapse-free survival and overall survival of TROJANlow/ZMYND8high and TROJANhigh/ZMYND8low patients in FUSCC cohorts 1 (D) and 2 (E). (F) Schematic diagram of TROJAN regulating breast cancer progression via ZMYND8. See also fig. S9.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaat9820/DC1

    Fig. S1. TROJAN properties.

    Fig. S2. Expression pattern of TROJAN.

    Fig. S3. TROJAN phenotype.

    Fig. S4. Construction of the TROJAN knockout cell line.

    Fig. S5. Potential therapeutic role of TROJAN in breast cancer progression.

    Fig. S6. TROJAN associates with the ZMYND8 protein.

    Fig. S7. TROJAN degrades the ZMYND8 protein.

    Fig. S8. TROJAN and ZMYND8 regulate the transcription of diverse target genes.

    Fig. S9. TROJAN regulates breast cancer progression via ZMYND8.

    Table S1. TNBC-related HERVs, basic patient information, primers, and MS data for proteins pulled down by TROJAN and SYSL1.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. TROJAN properties.
    • Fig. S2. Expression pattern of TROJAN.
    • Fig. S3. TROJAN phenotype.
    • Fig. S4. Construction of the TROJAN knockout cell line.
    • Fig. S5. Potential therapeutic role of TROJAN in breast cancer progression.
    • Fig. S6. TROJAN associates with the ZMYND8 protein.
    • Fig. S7. TROJAN degrades the ZMYND8 protein.
    • Fig. S8. TROJAN and ZMYND8 regulate the transcription of diverse target genes.
    • Fig. S9. TROJAN regulates breast cancer progression via ZMYND8.
    • Legend for Table S1

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). TNBC-related HERVs, basic patient information, primers, and MS data for proteins pulled down by TROJAN and SYSL1.

    Files in this Data Supplement:

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