Abstract
‘Gain’ of supernumerary copies of the 8q24.21 chromosomal region has been shown to be common in many human cancers1,2,3,4,5,6,7,8,9,10,11,12,13 and is associated with poor prognosis7,10,14. The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region and is consistently co-gained with an adjacent ‘gene desert’ of approximately 2 megabases that contains the long non-coding RNA gene PVT1, the CCDC26 gene candidate and the GSDMC gene. Whether low copy-number gain of one or more of these genes drives neoplasia is not known. Here we use chromosome engineering in mice to show that a single extra copy of either the Myc gene or the region encompassing Pvt1, Ccdc26 and Gsdmc fails to advance cancer measurably, whereas a single supernumerary segment encompassing all four genes successfully promotes cancer. Gain of PVT1 long non-coding RNA expression was required for high MYC protein levels in 8q24-amplified human cancer cells. PVT1 RNA and MYC protein expression correlated in primary human tumours, and copy number of PVT1 was co-increased in more than 98% of MYC-copy-increase cancers. Ablation of PVT1 from MYC-driven colon cancer line HCT116 diminished its tumorigenic potency. As MYC protein has been refractory to small-molecule inhibition, the dependence of high MYC protein levels on PVT1 long non-coding RNA provides a much needed therapeutic target.
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Change history
06 August 2014
Figure 2a, b, d scale bars were missing and have been added. Minor edits were made to the Fig. 2 legend.
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Acknowledgements
We thank A. T. Vogel for writing statistical analysis scripts; Research Animal Resources, University of Minnesota, for maintaining the mouse colony; S. Horn and L. Oseth for embryonic stem cell blastocyst injection and FISH analysis respectively. This work was supported by Masonic Cancer Center Laboratory start-up funds (to A.B.), and by grants from the Masonic Scholar Award (to A.B.), the Karen Wyckoff Rein in Sarcoma Fund (to A.B.), Translational Workgroup Pilot Project Awards by the Institute of Prostate and Urologic Cancer, University of Minnesota (to A.B.) and an American Cancer Society Institutional Research Grant (award 118198-IRG-58-001-52-IRG92, to A.B.). A.T. was supported by an Indo-US fellowship from the Indo-US Science and Technology Forum.
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Y.Y.T. and A.B. conceptualized the research programme and designed the experiments; Y.Y.T., B.S.M., H.K., A.T., R.A., P.R., B.R., K.G., T.C.B., J.E., Y.K. and A.B. performed the experiments. Y.Y.T. and W.G. analysed the data; M.G.O. and Y.Y.T. performed the histological analyses; K.L.S., D.A.L., Y.M., Y.K. and A.B. supervised experiments and data analysis; A.B. and Y.M. wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 2 Generating the mouse strains.
a–c, Chromosome engineering of Hprt-deficient mouse AB2.2 embryonic stem cells was used to develop mouse strains containing an extra copy of Myc (gain(Myc)) (a), Pvt1/Ccdc26/Gsdmc (gain(Pvt1,Ccdc26,Gsdmc)) (b) and Myc/Pvt1/Ccdc26/Gsdmc (gain(Myc,Pvt1,Ccdc26,Gsdmc)) (c). Gene targeting was performed in AB2.2 with targeting vectors obtained from the Mutagenic Insertion and Chromosome Engineering Resource (MICER) that were modified to facilitate detection of correctly targeted clones by PCR. These electroporated embryonic stem cells were cultured in G418 (G, 180 µg ml−1) or puromycine (P, 3 µg ml−1) for 7–10 days. Correctly targeted clones were identified by PCR analysis. Double-targeted embryonic stem cells were electroporated with the transient Cre-recombinase expression vector pOG231. After subsequent selection of recombinants by using hypoxanthine aminopterin thymidime (H) media for 7 days and then recovery of recombinants by using hypoxanthines and thymidine media for 2 days, the HRGRPR clones containing one mouse chromosome 15 with the targeted region duplication (dp) and the other mouse chromosome 15 with the targeted region deletion (df) were identified. d, Summary of gene targeting of mouse genome at the MycL, MycR and GsdmcR loci. e, FISH analysis of gain/loss Myc,Pvt1,Ccdc26,Gsdmc embryonic stem cells with balanced allele for the Myc/Pvt1/Ccdc26/Gsdmc region. Metaphase and interphase preparations from the engineered cells were probed with BAC clone specific for chromosome 15 and located outside (RP23-18H8, red) and within (RP24-78D24, green) the engineered region. The alleles containing the deletion and the duplication are marked.
Extended Data Figure 3 Histopathology of mammary adenocarcinomas and lung metastasis in gain(Myc,Pvt1, Ccdc26,Gsdmc), MMTVneu/+ and gain(Myc,Pvt1,Ccdc26,Gsdmc) respectively.
a, b, Mammary adenocarcinoma in gain(Myc,Pvt1,Ccdc26,Gsdmc),MMTVneu/+ mice. Representative histopathology of mammary tumours from gain(Myc,Pvt1,Ccdc26,Gsdmc),MMTVneu/+ mice. Image shows a solid, expansile tumour that is invading a small blood vessel (arrow; bar, 1000 µm) (a), and numerous mitotic figures (arrows; bar, 100 µm) (b). c, Summary of histopathology showing early onset of mammary adenocarcinoma in gain(Myc,Pvt1,Ccdc26,Gsdmc),MMTVneu/+ mice compared with MMTVneu/+ mice. d, Image of lung metastasis in gain(Myc,Pvt1,Ccdc26,Gsdmc) with spontaneous mammary adenocarcinoma (YYT 528,). e, Histopathology summary of spontaneous, low-penetrance tumour onset in gain(Myc,Pvt1,Ccdc26,Gsdmc) mice.
Extended Data Figure 4 Abnormal oncogenic stress, proliferation and differentiation in gain(Myc,Pvt1,Ccdc26, Gsdmc) mammary ducts.
a, b, Western blot analysis of p53 (a) and phospho-Erk1/2 (b) in total protein lysates from mammary glands of indicated genotypes. The relative densities of p53 and p-ERK1/2 were calculated by normalizing against the GAPDH and total ERK1/2 protein levels respectively. c, Immunofluorescence analysis of ERα (green) on sections of mammary ducts. Cell nuclei positive for ERα are presented as the percentage of total epithelial cell nuclei (DAPI, blue). Images shown are representative three mice per genotype. d, Haematoxylin and eosin staining of the mammary ducts from wild type and gain (M,P,C,G) mice showed precocious alveolar-like phenotype in the latter. This aberrant structure is shown at higher magnification in the right row. e, Immunofluorescence co-staining for DAPI (blue), luminal marker K8 (red) and myoepithelial marker K14 (green) in mice. Arrowheads indicate co-expression of K8 and K14. DAPI-stained nuclei in blue. Mean ± s.e.m. for a–c (n = 3). *P < 0.05, ***P < 0.001 by two-tailed Student’s t-test; error bars, s.e.m.
Extended Data Figure 5 Gasdermin expression in mouse mammary tissues.
a, Gsdmc is not expressed in mouse mammary tissue. Semi-quantitative RT–PCR of Gsdmc transcript in mouse colon and mammary tissues. PCR was performed using equal amount of cDNAs derived from colon and mammary tissues, for cycles as indicated. –RT indicates samples treated without reverse transcriptase. β-actin used as a loading control. NC, negative control (water). b–d, Representative RT–qPCR analysis of Gsdmc2 (b), Gsdmc3 (c) and Gsdmc4 (d) mRNA in 10-week-old virgin mouse mammary tissues of all genotypes. Mean ± s.e.m. for b–d (n = 3); error bars, s.e.m.
Extended Data Figure 6 PVT1 depletion results into reduction in proliferation in SK-BR-3 and MDA-MB-231 breast cancer cell lines.
a, b, Proliferation assay of human breast cancer cell lines SK-BR-3 (a) and MDA-MB-231 (b) growing in three-dimensional culture after the cell lines were transfected with siCtrl, siMYC, siPVT1 and both (siMYC + siPVT1). Transfection efficiency in each cell line was confirmed as mentioned in the text. Mean ± s.e.m. for a and b (n = 3). **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test. c, d, Inhibition of miRNA expression coded by the PVT1 locus shows no loss of proliferation in SK-BR-3 cells. SK-BR-3 cells transfected with antisense miRNAs were grown in three-dimensional culture as described before. c, Relative expression of individual miRNA in cells treated with its corresponding inhibitor, normalized to the control experiments Each data point represents the mean ± s.e.m. (n = 3, except for miR-1207, where n = 6). d, Percentage of Ki-67 positive cells denotes the proliferation index. Bar graph, mean ± s.e.m. (n = 3); error bars, s.e.m.
Extended Data Figure 7 PVT1 regulates MYC protein level in MDA-MB-231 breast cancer cells.
a, RT–qPCR measurement of MYC (left) and PVT1 (right) RNA levels in MDA-MB-231 cells 48 h after transfection with the indicated siRNAs. b, Reduction in MYC protein in PVT1-depleted MDA-MB-231. Western blot analysis for MYC protein in the total lysates obtained from the MDA-MB-231 cell line transfected with different siRNAs. The relative density for each category was determined by normalizing against the intensity of the GAPDH band. c, Stability assay for MYC protein in MDA-MB-231 cells. Cells were transfected with siCtrl and siPVT1 and then treated with 10 μM cycloheximide for different time points (top panel). The relative density was determined by comparing against the GAPDH level (bottom panel). Mean ± s.e.m. for a–c (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test. Error bars, s.e.m.
Extended Data Figure 8 PVT1 and MYC co-localize in the SK-BR-3 nuclei.
a, Specificity of fluorescent-labelled anti-PVT1 RNA probe in SK-BR-3 cells. Fluorescent images of SK-BR-3 cells treated with no probe, sense and anti-sense PVT1 RNA probe. DAPI (blue) stain is shown in the lower panel. b, c, Expression and co-localization of MYC and PVT1 in SK-BR-3 nuclei. Representative fluorescent images of SK-BR-3 cells treated with fluorescently labelled anti-MYC antibody (green) and anti-PVT1 RNA (magenta) (b). DAPI indicating cell nuclei is shown in blue. Cells expressing MYC and PVT1 are indicated by red arrows whereas those expressing low levels of MYC and PVT1 are indicated by white arrows. Merge panel represents overlapping images of DAPI, MYC and PVT1 panels. The nuclei with co-localization of MYC and PVT1 are shown by red arrows. c, Quantification of SK-BR-3 nuclei with co-localization of MYC and PVT1 (n = 87).
Extended Data Figure 9 Incidence MYC and PVT1 co-gain in human cancers.
a, Number of 8q24 gain-associated human cancers showing gain of MYC but not PVT1 (blue), gain of PVT1 but not MYC (orange) and co-gain of MYC + PVT1 (green) in TCGA (left) and Progenetix copy-number database (right). b, Pie chart showing breast cancer samples in TCGA expressing high HER2 transcript with or without co-gain of MYC + PVT1. c, The ratio of MYC + PVT1 + CCDC26 and MYC + PVT1 + GSDMC co-amplification among MYC + PVT1 co-gained TCGA samples at different amplification levels (segment mean cutoff). d, The ratio of MYC + PVT1 co-amplification among MYC-gained cancers on different segment mean cutoffs. e, f, Tissue microarray analysis of PVT1 RNA and MYC protein expression in primary human tumours. Images of 32 primary human tumours showing in situ hybridization using digoxigenin-labelled PVT1 probe (purple, top panel) and MYC immunohistochemistry using anti-MYC antibody (brown, bottom panel) (e). f, Specification of multiple organ normal and diseased tissue microarray, single core per case, eight types of tumour (breast, colon, oesophagus, kidney, liver, lung, rectum, stomach) (BC00119).
Extended Data Figure 10 Generating the ΔPVT1 HCT116.
a, Schematic representation of CRISPR-mediated deletion of 307 kilobase PVT1 gene in HCT116. Black triangles denote CRISPR specific for upstream of exon 1 and downstream of exon 8 of PVT1. PCR primers F1, F2 and R1 are denoted. b, A CRISPR-mediated deletion of PVT1 (ΔPVT1) can be detected by PCR amplicon using primers F1 and R1, whereas the control with PVT1 locus intact (PVT1+) can be screened by using F2 and R1. c, RT–qPCR analysis of PVT1 transcript in PVT1+ and ΔPVT1 HCT116 cells (n = 3). d, Relative cellular proliferation abilities of PVT1+ and ΔPVT1 HCT116 cells were evaluated by MTS assays. Data are mean ± s.e.m. for c and d (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test. Error bars, s.e.m. e, Luciferase-based images of the presence of tumour lesions detected 3–17 days after subcutaneous implantation of PVT1+ and ΔPVT1 HCT116 cells in nude mice.
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Tseng, YY., Moriarity, B., Gong, W. et al. PVT1 dependence in cancer with MYC copy-number increase. Nature 512, 82–86 (2014). https://doi.org/10.1038/nature13311
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DOI: https://doi.org/10.1038/nature13311
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