Identification of a chromosome 11 amplicon as the major genomic alteration in MMTV-Myc mammary tumors

We performed comparative genomic hybridization (CGH) of mammary gland tumors from eight genetically engineered mouse models to identify genomic loci containing genes with altered expression that potentially cooperate with oncogenes or suppressor genes in promoting tumorigenesis. On average, DNAs from 5–6 non-necrotic tumor samples from each model were analyzed by array CGH using the Agilent 44K array platform. Spleen DNA from the background strain served as the corresponding control. Our CGH results identified previously reported copy number variants (CNVs) and chromosomal aberrations in these models [8, 13–16], validating the results. In addition to the amplification of distal chromosome 6 in C3(1)-Tag model that we had previously reported [17], we identified amplification of chromosome 6 in four additional tumor models, gain of partial or whole chromosome 15 in four models, and loss of chromosome 4 in some C3(1)-Tag-driven tumors (Fig. 1), consistent with prior reports [8, 13, 15, 16, 18]. Interestingly, 80 % of MMTV-Myc-driven tumors exhibited minimal genomic changes except for amplification of the distal part of chromosome 11. We also observed this amplification in some MMTV-PyMT and BRCA−/−; p53+/− tumors as have been previously reported [8, 13, 15, 16]. However, these two models also exhibited other large regions of chromosomal amplifications. Therefore, we chose to focus on the MMTV-Myc model since the chromosome 11 amplification region likely contains genes required for Myc tumorigenesis. Importantly, this region is syntenic to human chromosome 17q23-qter, which is often amplified in human breast cancer patients [19–22].

Fig. 1 DNA copy number analysis. Array CGH analysis of mouse mammary gland tumors from eight genetically engineered models of breast cancer. 5–6 tumors were used for each model. The threshold line is drawn at 35 % of samples. Genomic regions of significant gains are shown in blue and significant losses are shown in red. Arrow indicates the amplification of distal mouse chromosome 11 Full size image

Identification of genes overexpressed in the chromosome 11 amplicon in MMTV-Myc mammary tumors

To gain more insight into the function of the genes located in this region, we determined the minimal region that was amplified in chromosome 11 across the MMTV-Myc tumors and found that it contained 246 genes and one microRNA. The exact chromosomal coordinates for those genes are presented in Additional file 1: Figure S1. To identify the genes that had higher expression in MMTV-Myc tumors compared to normal mammary gland tissue or tumors from other models that did not contain the chromosome 11 amplification, we performed microarray analysis using RNA from normal glands as well as several mammary gland tumors derived from MMTV-HRas and MMTV-Her2/Neu mice (Fig. 2). Using these data together with a literature-based screen of known gene functions, we selected seven genes from the chromosome 11 candidate interval for further validation (FBF1, Ube2o, TK1, Birc5, Sumo2, Tnrc6c, and JMJD6).

Fig. 2 Gene expression microarray analysis for chromosome 11 amplified region. The heatmap shows the differential gene expression in mammary gland tumors from MMTV-Myc transgenic mice with chromosome 11 amplification versus MMTV-Her2, or MMTV-HRas tumors lacking the chromosome 11 amplification, or normal lactating mammary glands from FVB/N mice. Genes labeled in red are expressed at higher than median levels and genes labeled in green are expressed at lower than median levels. Genes selected for further validation are indicated on the left side Full size image

To identify potential candidate cell lines derived from MMTV-Myc tumors for further in vitro studies, we performed CGH analysis of several cell lines and found that the Myc83 cell line harbors the chromosome 11 amplicon, as previously observed [23], while the 88CT1 cell line has relatively few CNVs without amplification of the chromosome 11 locus (Additional file 1: Figure S2A). We also found increased expression levels of selected genes (JMJD6, Tnrc6c, and Ube2o) by RT-qPCR in the Myc83 cells compared to 88CT1 cells, consistent with the status of the chromosome 11 amplification (Additional file 1: Figure S2B).

Identification of JMJD6 as a gene that suppresses Myc-induced apoptosis

Since Myc expression increases apoptosis in primary cells, which is a major response preventing full transformation of cells by Myc alone, we tested the hypothesis that the increased expression of candidate genes would suppress Myc-induced cell death, whereas depletion of any of the seven selected genes would increase Myc-induced cell death in vitro. Myc-induced apoptosis in many cases requires an intact p53 pathway. Sequence analysis confirmed the wild-type status of p53 in both Myc83 and 88CT1 cells. However, etoposide treatment of these cell lines revealed that Myc83 responded to etoposide with a robust increase in p53 and p21 protein levels, while 88CT1 had a much lower expression of p53 (possibly because of the amplification of MDM2 in these cells, as determined by CGH analysis). Therefore, for the primary apoptosis screen, we chose Myc83 cells that express high constitutive levels of c-Myc and contain the chromosome 11 amplification. The limitation of this model is that one cannot conclude that cell death is Myc-dependent or whether the tested genes have a more general effect on cell viability.

To clarify this question, we established another model using normal murine mammary gland (NMuMG) epithelial cells with inducible expression of MycER™ where c-Myc is fused in frame with the mutated estrogen receptor-binding domain which makes it refractory to beta-estradiol but that can be activated by the addition of 4-hydroxytamoxifen [24]. First, we tested five different small hairpin RNA (shRNA) constructs for each gene and selected those that provided at least a 50 % reduction in gene expression. These shRNAs were then stably expressed in Myc83 cells and NMuMG-MycERTM cells and the resultant cells were treated with etoposide or glucose deprivation. As shown in Fig. 3 and Additional file 1: Figure S3, the most consistent increase in Myc-dependent cell death in both cell types was obtained after depletion of JMJD6. The efficiency of JMJD6 knock-down by two shRNAs is shown in Additional file 1: Figure S4. Several other genes showed promising results in cooperation with Myc in MNuMG cells (FBF1) and others in Myc83 cells (Sumo2), which may reflect limited cell type-specific functions of these genes. We, therefore, focused on exploring the cooperation of JMJD6 with c-Myc in cellular transformation, tumor progression, and metastases.

Fig. 3 Analysis of cell death induced by glucose deprivation or etoposide treatment in cells with JMJD6 knock-down. a Myc83-derived cell lines (parental line from a MMTV-Myc tumor) with stable expression of two independent shRNAs targeting JMJD6 or with empty vector (EV) control were treated with 100 μM etoposide or grown in glucose-free media for 20 h. Cell death was measured using CytoTox-Glo reagent. The experiments were repeated 3 times and the percentage of dead cells in each experiment was expressed relative to control (EV) cells. b NMuMG cells with MycER™ and shJMJD6 expression were first treated with 150 nM 4-OHT (MycON) or ethanol (MycOFF) for 24 h to activate Myc and then treated as in a Black bars—MycOFF (ethanol-treated cells), open bars—MycON (4-OHT-treated cells). Results were normalized to EV control with MycOFF. *p < 0.05, **p < 0.01 Full size image

The anti-apoptosis effect of JMJD6 is dependent upon JMJD6 enzymatic activity

JMJD6 is an enzyme with pleiotropic functions that has been recently implicated in the breast, and some other cancers where high expression of JMJD6 was an indicator of poor prognosis [25–28]. We further validated our model system using NMuMG cells with constitutive overexpression of JMJD6 and an inducible c-Myc. As expected, ectopically expressed JMJD6 was localized to cell nuclei whereas control cells exhibited ectopic cytoplasmic expression of LacZ (Additional file 1: Figure S5A). The overall levels of JMJD6 in transfected cells were close to physiological levels observed in cells with amplified chromosome 11 (about 3-fold over control cells, Additional file 1: Figure S5B). Cells expressing high levels of c-Myc proved to be sensitive to multiple stress conditions, including depletion of nutrients or growth factors, or treatment with DNA-damaging agents, leading to cell death. C-Myc induction in NMuMG cells followed by exposure to four different stress conditions resulted in a significant increase in cell death, which was reduced by co-expression with JMJD6 (Fig. 4a–d).

Fig. 4 Overexpression of wild-type, but not mutated, JMJD6 suppresses Myc-induced cell death in response to different stress conditions. NMuMG cells stably expressing MycERTM or empty pBabe vector were transduced with JMJD6-V5 or LacZ-V5 control. Cells were treated with 4-OHT or ethanol as in Fig. 3 and placed in glucose-free (a), serum-free (b), or glutamine-free (c) media or treated with etoposide (d) for another 20 h. Cell death was measured as in Fig. 3. Black bars—MycOFF, open bars—MycON. e Catalytically inactive JMJD6H187A (JMJD6mut) is not able to suppress Myc-induced cell death. *p < 0.05, **p < 0.01. f Western blot analysis shows equal ectopic expression of JMJD6 or mutated JMJD6 in NMuMG cells with our without Myc expression Full size image

JMJD6 is an iron- and 2-oxoglutarate-dependent dioxygenase with the capability to hydroxylate lysine residues in histone and non-histone proteins [26, 29, 30]. Lysine hydroxylation of RNA splicing factors results in production of differentially spliced pre-messenger RNA (mRNA) molecules [31], while hydroxylation of histone proteins may result in changes in transcriptional regulation of targeted gene expression [32]. In order to determine whether the enzymatic activity of JMJD6 is necessary for the inhibition of Myc-induced cell death, we mutated His187 to Ala in the iron-coordinating center of the enzyme that has previously been showed to inactivate JMJD6 and demonstrated that mutant JMJD6 does not suppress cell death (Fig. 4e). Western blotting confirmed that the levels of mutated protein expression were similar to the expression of the wild-type JMJD6 (Fig. 4f).

To further prove that high levels of wild-type JMJD6, but not its mutated form, inhibit cell death, we examined protein levels of cleaved caspase 3 and PARP (markers of apoptosis) following Myc induction and exposure to different stress conditions. While control cells showed robust cleavage of both enzymes upon c-Myc induction, cleavage was clearly diminished when cells co-expressed wild-type JMJD6 (Fig. 5). However, the expression of mutant JMJD6 had a minimal effect on suppressing caspase 3 and PARP cleavage, which remained similar to levels observed in control cells (Fig. 5).

Fig. 5 Western blot analysis of apoptotic markers in Myc-induced cells in the presence of wild-type or mutated JMJD6. NMuMG cells were treated with 150 nM 4-OHT to induce Myc or ethanol and placed in glucose- or glutamine-free media or treated with 100 mM of etoposide. Expression of cleaved PARP and cleaved caspase 3 was determined by Western blot demonstrating reduced levels in the presence of wild-type, but not mutant, JMJD6 Full size image

In order to understand mechanisms by which JMJD6 inhibits Myc-induced cell death, we first analyzed its effect on Myc protein levels and cellular localization. Additional file 1: Figure S5A shows that MycER™ or endogenous levels of c-Myc were not compromised by overexpression of JMJD6. Also, in the presence of 4-hydroxytamoxifen, ectopically expressed MycER™ re-localizes to the cell nucleus similar to control cells without JMJD6 overexpression (Additional file 1: Figure S6B).

Identification of mechanisms contributing to JMJD6 inhibition of Myc-induced apoptosis

We then sought to establish which pathways involved in Myc-induced cell death are inhibited by elevated levels of JMJD6 expression. In many cellular models, Myc-induced apoptosis stimulates induction of p19ARF that sequesters MDM2 ubiquitin ligase, allowing for the upregulation of p53 that activates apoptotic pathways [33–35]. We first found that in NMuMG cells, JMJD6 downregulates the endogenous levels of p19ARF and p53, with or without c-Myc induction (Fig. 6a). It has been known that p19ARF may be suppressed by Bmi1 protein; however, we found no difference in Bmi1 protein levels in cells with high expression of JMJD6 (Fig. 6a). We also analyzed p19ARF and p53 induction after treatment with the DNA-damaging agent etoposide in the presence of JMJD6. JMJD6 expression resulted in lower levels of p19ARF, p53 total protein, and its Ser18 phosphorylated form as well as p21 proteins at all time points after etoposide treatment, compared to LacZ control (Fig. 6b).

Fig. 6 JMJD6 overexpression reduces p53 and p19ARF levels. a Immunoblotting of NMuMG cells with ectopic expression of JMJD6 or LacZ control shows decreased protein levels of p53 and p19ARF in cells with or without Myc induction. b Cells were stimulated with 4-OHT to induce Myc and then treated with 100 mM etoposide for 4 and 8 h to analyze p53 induction. Immunoblots show lower levels of p53 or Ser18-phosphorylated p53 as well as p19ARF and p21 in cells expressing JMJD6. Intensity of each band normalized to β-actin was obtained using ImageJ software. c Overexpression of p19ARF in the presence of JMJD6 restores Myc-induced cell death. NMuMG cells expressing MycER™ together with JMJD6, LacZ, or JMJD6 plus p19ARF were treated with 4-OHT (MycON) or ethanol (MycOFF) for 24 h and placed in serum-free media for another 24 h. Percentage of dead cells was measured with CytoTox-Glo reagent. Black bars—MycON, open bars—MycOFF. d Western blot analysis of NMuMG cells with ectopic overexpression of p19ARF. e RT-qPCR analysis shows that overexpression of JMJD6 in NMuMG cells inhibits p19ARF transcription. Black bars—NMuMG cells expressing LacZ control, open bars—cells expressing wild-type JMJD6. f Knock-down of endogenous JMJD6 with three different shRNAs increases mRNA levels of p19ARF Full size image

Our finding that JMJD6 may reduce p19ARF stimulation by Myc led us to determine whether the blunting effect of JMJD6 on Myc-induced cell death is p19ARF dependent. We overexpressed p19ARF protein in cells with JMJD6 and the inducible MycERTM and determined levels of cell death after starving cells in serum-free conditions. Indeed, increased levels of p19ARF protein rescued Myc-induced apoptosis in cells with high JMJD6 expression, demonstrating that p19ARF suppression by JMJD6 contributes to the alleviation of cell death (Fig. 6c, d).

To further confirm that high expression of JMJD6 would correlate with decreased expression of p19ARF in vivo, we analyzed expression of these two genes in mouse MMTV-Myc mammary gland tumors. This experiment also showed robust negative correlation between JMJD6 and p19ARF (Additional file 1: Figure S7).

JMJD6 reduces p19ARF expression through histone H4 modifications on the p19ARF promoter region

JMJD6 is involved in post-translational modifications of non-histone and histone proteins [29, 30, 32, 36]. However, when JMJD6 regulates histone modifications, it modifies mRNA levels of the target gene. To discriminate which of the pathways is involved in p19ARF silencing by JMJD6, we studied p19ARF transcript levels in the presence of high JMJD6 expression. We clearly observed that JMJD6 overexpression significantly reduces mRNA levels of p19ARF (Fig. 6e), while three different JMJD6 shRNA constructs significantly increased p19ARF mRNA when expressed in NMuMG cells (Fig. 6f).

Previously published data suggest that JMJD6 is able to remove methyl groups from symmetrically (H4R3me2s) or asymmetrically (H4R3me2a) methylated arginines [32, 36]. This allows JMJD6 to act as a transcriptional activator or repressor, as H4R3me2s modification is associated with repressed chromatin [37, 38] and H4R3me2a is a mark for transcriptionally active chromatin [37, 39, 40]. To this end, we performed chromatin immunoprecipitation experiments to compare the amounts of activating H4R3me2a associated with the p19ARF promoter in cells expressing high levels of JMJD6 versus cells with low expression of JMJD6. First, we found that control cells expressing LacZ or mutated JMJD6 have a significantly higher presence of H4R3me2a within the p19ARF promoter than cells with overexpression of JMJD6 (p = 0.01) (Fig. 7a). Furthermore, chromatin immunoprecipitation with antibodies specific for JMJD6 showed that JMJD6 as well as mutated JMJD6 protein is bound to the p19ARF promoter (Fig. 7b). We also analyzed JMJD6 effect on p16 and found no expression of p16 in control or JMJD6-overexpressing NMuMG cells by RT-qPCR or Western blotting. Therefore, to prove specificity of anti-JMJD6 antibody in ChIP assay, we used p16 promoter primers and found no binding of JMJD6 to p16 promoter (Additional file 1: Figure S8). In addition, since control LacZ, JMJD6, and JMJD6 mutants were fused with C-terminal V5 tag, we performed ChIP analysis using V5-conjugated agarose and confirmed higher abundance of JMJD6 and JMJD6 mutant binding to the p19ARF promoter compared to LacZ control (Fig. 7c).

Fig. 7 JMJD6 binds to the p19ARF promoter and decreases histone H4R3 asymmetric dimethylation of the p19ARF promoter. ChIP analysis of the p19ARF promoter in NMuMG cells expressing LacZ, JMJD6, or JMJD6 mutant using immunoprecipitation with a anti-H4R4me2a, b anti-JMJD6 antibody, c V5-conjugated agarose. d ChIP analysis of HeLa cells shows binding of endogenous JMJD6 to the human p14ARF promoter. Two different primer sets (distal and proximal) were used for qPCR after immunoprecipitation. Primers for human β-globin gene promoter were used as a positive control Full size image

Since endogenous mouse JMJD6 is poorly recognized by the antibody available for ChIP studies, we decided to test human HeLa cells to analyze whether endogenous JMJD6 in these cells is found on the ARF promoter. We tested the human p14ARF promoter with two primer sets using a human JMJD6 antibody for ChIP and found higher levels of JMJD6 associated with the ARF promoter compared to immunoprecipitation with a non-specific IgG (Fig. 7d). In summary, we found that in non-tumorigenic mouse epithelial cells, JMJD6 may collaborate with c-Myc to initiate tumor formation by suppressing Myc-induced cell death by inhibiting transcriptional expression of the p19ARF tumor suppressor protein.

JMJD6 promotes Myc-induced tumor formation

We sought to determine to what extent established mammary tumors remain dependent on JMJD6 expression for tumor maintenance and an aggressive phenotype. We first knocked down JMJD6 in Myc83 cells with elevated JMJD6 expression using two different shRNAs and found a significant delay in tumor formation compared to control cells when injected into mouse mammary fat pad (Fig. 8a). To show the reciprocal effect of increased JMJD6 expression in low JMJD6-expressing cells, overexpression of JMJD6 in 88CT1 cells resulted in significantly larger tumors compared to LacZ control-expressing cells (Fig. 8b). The tumors derived from 88CT1 cells ectopically expressing JMJD6 exhibited significantly less apoptosis by TUNEL assay (Fig. 8c) than control cells but no difference in expression of the cell proliferation marker Ki67 (Fig. 8d).

Fig. 8 JMJD6 promotes tumor growth of cells derived from MMTV-Myc mammary gland tumors. a 106 Myc83 cells (with amplification of the chromosome 11 locus containing JMJD6) stably expressing empty vector (EV) or two independent shRNAs targeting JMJD6 were injected into mammary fat pads of FVB/N mice and tumors were measured over the next month. b 5 × 105 88CT1 cells (no chromosome 11 amplification) with stable expression of LacZ control or wild-type JMJD6 were surgically implanted into mammary fat pads and tumor growth was measured over the next 20 days. c Tumors from b were formalin fixed and sectioned and the percentage of dead cells was analyzed by TUNEL assay. d Tumors from b were immunostained with anti-Ki67 antibodies and signal intensity was quantitated by ImageJ software. e Western blot analysis of pro-survival genes in 88CT1 cells expressing JMJD6 Full size image

Interestingly, we demonstrated that 88CT1 cells established from MMTV mice have a compromised p53 pathway, possibly resulting from amplification of MDM2 (gain of 8 copies) as determined by array CGH. We found that the reduction in the percentage of apoptotic cells in 88CT1 cells overexpressing JMJD6 was associated with elevated levels of the Bcl2 anti-apoptotic family members Bcl-xl and Bcl-w (Fig. 8e) that were previously shown to inhibit Myc-induced cell death [41–43].

JMJD6 enhances cell migration, invasion, and metastases

MMTV-Myc mammary gland tumors usually produce well-differentiated tumors with a relatively long latency and relatively few metastases [12, 44, 45], implying that additional genetic alterations are required for a more aggressive phenotype. However, several observations raise the possibility that elevated expression of Myc protein may inhibit cellular migration, invasion, and metastasis formation in mouse xenograft models of breast cancer [46]. We therefore investigated whether JMJD6 might enhance migration, invasion, and metastases of MMTV-Myc mammary tumor cells.

Ectopic expression of JMJD6 in 88CT1 cells resulted in a 2–3-fold increase in motility and invasion compared to non-JMJD6-expressing cells using the Boyden chamber assays (Fig. 9a, b) and was associated with increased expression of the EMT markers Snail and Twist1, while others, vimentin and Slug, remained unchanged (Fig. 9c). To test the metastatic propensity of 88CT1 cells overexpressing JMJD6, we injected those cells or LacZ control cells via mouse tail vein and analyzed lung sections 21 days later. Control cells produced very few and in many cases no metastatic nodules while cells with elevated expression of JMJD6 showed a dramatic 20-fold increase in the number of lung colonies (p < 0.0001, Fig. 9d, e). These results demonstrate that JMJD6 contributes to Myc-induced mammary gland tumor maintenance and confers a highly metastatic tumor phenotype.

Fig. 9 JMJD6 increases the metastatic propensity of c-Myc-expressing cells. Transwell migration (a) and invasion (b) assays of 88CT1 cells overexpressing JMJD6 compared to LacZ control (*p < 0.05, **p < 0.01, respectively). c Western blot analysis of EMT markers in the cells used in a and b shows increased expression of Snail and Twist1 in cells with high expression of JMJD6. d Lung colonization in vivo. 5 × 105 cells used in a and b were injected by tail vein into FVB/N mice. After 20 days, the lungs were formalin fixed and stained with H&E. e Quantitation of metastatic nodules per lung of mice shown in d Full size image

High expression of JMJD6 in human breast tumors is associated with a worse prognosis of Myc-high tumors

The in vitro and in vivo results presented above suggest that the combination of high expression of Myc and JMJD6 might be associated with a poor clinical prognosis. To determine whether this might be true for human breast cancers, we performed an in silico analysis using the METABRIC database that contains microarray data and clinical information on about 2000 breast cancer patients. All cases were divided into four subsets, consisting of high/low, high/high, low/high, and low/low of JMJD6/Myc expression. When we compared patients with high versus low JMJD6 expression, we observed a highly significant effect in samples with high Myc rather than low Myc expression (compare p values for left and right panels in Fig. 10a, c). We determined that high JMJD6 expression is associated with a poor prognosis for ER-positive breast cancer patients and not for ER-negative breast cancer (Fig. 10b), consistent with a previous report analyzing JMJD6 expression as a biomarker for poor prognosis in ER+ breast cancer [25]. Overall, this analysis predicts that JMJD6 gene expression may be a discriminating factor for survival of patients with high Myc expression in ER-positive breast cancer patients.