A RUNX2-Mediated Epigenetic Regulation of the Survival of p53 Defective Cancer Cells.

The inactivation of p53 creates a major challenge for inducing apoptosis in cancer cells. An attractive strategy is to identify and subsequently target the survival signals in p53 defective cancer cells. Here we uncover a RUNX2-mediated survival signal in p53 defective cancer cells. The inhibition of this signal induces apoptosis in cancer cells but not non-transformed cells. Using the CRISPR technology, we demonstrate that p53 loss enhances the apoptosis caused by RUNX2 knockdown. Mechanistically, RUNX2 provides the survival signal partially through inducing MYC transcription. Cancer cells have high levels of activating histone marks on the MYC locus and concomitant high MYC expression. RUNX2 knockdown decreases the levels of these histone modifications and the recruitment of the Menin/MLL1 (mixed lineage leukemia 1) complex to the MYC locus. Two inhibitors of the Menin/MLL1 complex induce apoptosis in p53 defective cancer cells. Together, we identify a RUNX2-mediated epigenetic mechanism of the survival of p53 defective cancer cells and provide a proof-of-principle that the inhibition of this epigenetic axis is a promising strategy to kill p53 defective cancer cells

β-Catenin Does Not Confer Tumorigenicity When Introduced into Partially Transformed Human Mesenchymal Stem Cells

Although osteosarcoma is the most common primary malignant bone tumor in children and adolescents, its cell of origin and the genetic alterations are unclear. Previous studies have shown that serially introducing hTERT, SV40 large TAg, and H-Ras transforms human mesenchymal stem cells into two distinct sarcomas cell populations, but they do not form osteoid. In this study, β-catenin was introduced into mesenchymal stem cells already containing hTERT and SV40 large TAg to analyze if this resulted in a model which more closely recapitulated osteosarcoma. Results. Regardless of the level of induced β-catenin expression in the stable transfectants, there were no marked differences induced in their phenotype or invasion and migration capacity. Perhaps more importantly, none of them formed tumors when injected into immunocompromised mice. Moreover, the resulting transformed cells could be induced to osteogenic and chondrogenic differentiation but not to adipogenic differentiation. Conclusions. β-catenin, although fostering osteogenic differentiation, does not induce the malignant features and tumorigenicity conveyed by oncogenic H-RAS when introduced into partly transformed mesenchymal stem cells. This may have implications for the role of β-catenin in osteosarcoma pathogenesis. It also may suggest that adipogenesis is an earlier branch point than osteogenesis and chondrogenesis in normal mesenchymal differentiation

The effect of bone morphogenetic protein-2 on osteosarcoma metastasis.

PURPOSE:Bone Morphogenetic Protein-2 (BMP-2) may offer the potential to enhance allograft-host osseous union in limb-salvage surgery following osteosarcoma resection. However, there is concern regarding the effect of locally applied BMP-2 on tumor recurrence and metastasis. The purpose of this project was to evaluate the effect of exogenous BMP-2 on osteosarcoma migration and invasion across a panel of tumor cell lines in vitro and to characterize the effect of BMP-2 on pulmonary osteosarcoma metastasis within a xenograft model. EXPERIMENTAL DESIGN:The effect of BMP-2 on in vitro tumor growth and development was assessed across multiple standard and patient-derived xenograft osteosarcoma cell lines. Tumor migration capacity, invasion, and cell proliferation were characterized. In addition, the effect on metastasis was measured using a xenograft model following tail-vein injection. The effect of exogenous BMP-2 on the development of metastases was measured following both single and multiple BMP-2 administrations. RESULTS:There was no significant difference in migration capacity, invasion, or cell proliferation between the BMP-2 treated and the untreated osteosarcoma cell lines. There was no significant difference in pulmonary metastases between either the single-dose or multi-dose BMP-2 treated animals and the untreated control animals. CONCLUSIONS:In the model systems tested, the addition of BMP-2 does not increase osteosarcoma proliferation, migration, invasion, or metastasis to the lungs

RUNX2 has a pro-survival function in OS cells but not in MSCs.

<p>(<b>A</b>) I.B. to detect RUNX2, cleaved PARP and Tubulin in U2OS cells transduced with lentiviruses expressing shLuc, shRUNX2_3 or shRUNX2_4. (<b>B</b>) Cumulative cell numbers of U2OS cells transduced with lentiviruses expressing shLuc, shRUNX2_3 or shRUNX2_4. (<b>C</b>) I.B. to detect RUNX2, cleaved PARP and Tubulin in HOS-MNNG cells transduced with lentiviruses expressing shLuc, shRUNX2_3 or shRUNX2_4. (<b>D</b>) Cumulative cell numbers of HOS-MNNG cells transduced with lentiviruses expressing shLuc, shRUNX2_3 or shRUNX2_4. (<b>E</b>) I.B. to detect RUNX2, cleaved caspase3 and Tubulin in hMSC cells transduced with lentiviruses expressing shLuc, shRUNX2_3 or shRUNX2_4. (<b>F</b>) Cumulative cell numbers of hMSC cells transduced with lentiviruses expressing shLuc, shRUNX2_3 or shRUNX2_4. (<b>G</b>) mRNA levels of RUNX2 in hMSCs and four different human OS cell lines. (<b>H</b>) Percentage of apoptotic cells of hMSCs and four different human OS cell lines 6 days after transduced with lentiviruses expressing 50/50 of shRUNX2_3 and shRUNX2_4. (<b>I</b>) I.B. to detect Runx2, cleaved caspase 3, β-actin in Dunn (mouse OS cells), mMSC, and MC3T3_E1 cells transduced with lentiviruses expressing shLuc, shRunx2_4 or shRunx2_5. (<b>J</b>) Annexin V staining of Dunn (mouse OS cells), mMSC, and MC3T3_E1 cells transduced with lentiviruses expressing shLuc, shRunx2_4 or shRunx2_5 for 4 days.</p

Menin inhibitors induce the apoptosis in OS cells.

<p>(<b>A</b>) Immunoprecipitation (IP) showing the effect of Mi-2, Mi-3, and Mi-nc on the interaction between Menin and MLL1. (<b>B</b>) Flow cytometry and (<b>C</b>) W.B. showing that Mi-2 and Mi-3 induce the apoptosis of SAOS2 cells. (<b>D</b>) A model of a RUNX2-mediated epigenetic mechanism that regulates MYC expression and the survival of OS cells.</p

RUNX2 binding partner, CBFB, is required for OS cell survival.

<p>(<b>A</b>) Silver staining of FLAG-RUNX2 pulldown in SAOS cells. (<b>B</b>) I.B. to validate the interaction between CBFB and RUNX2 in SAOS2 cells. (<b>C</b>) Endogenous co-immunoprecipitation (Co-IP) assay of RUNX2 and CBFB in SAOS2 cells. (<b>D</b>) I.B. to detect CBFB in hMSCs and OS cells. (<b>E</b>) Realtime PCR to detect RNA levels of CBFB in hMSCs and OS cells. (<b>F</b>) I.B. to detect CBFB, cleaved caspase 3, and β-actin in SAOS2 cells transduced with lentiviruses expressing shLuc, shCBFB_1 or shCBFB_2. (<b>G</b>) Cumulative cell number of SAOS2 cells transduced with lentiviruses expressing shLuc, shCBFB_1 or shCBFB_2. (<b>H</b>) I.B. to detect CBFB, cleaved caspase 3, and β-actin in Hu09-M112 cells transduced with lentiviruses expressing shLuc, shCBFB_1 or shCBFB_2. (<b>I</b>) Cumulative cell number of Hu09-M112 cells transduced with lentiviruses expressing shLuc, shCBFB_1 or shCBFB_2. Error bars are SEM; t-test, **, p<0.01; *, p<0.05.</p

RUNX2 and CBFB co-occupy many loci in the genome.

<p>(<b>A</b>) Genomic views of RUNX2 and CBFB binding on the <i>SP7</i> and <i>ALPL</i> loci. Black bars underneath genomic view are identified peaks by the MACS algorithm. (<b>B</b>) Venn diagram of the numbers of RUNX2 peaks and CBFB peaks. (<b>C</b>) Spearman correlation of peaks intensity of 6022 overlapping RUNX2 and CBFB peaks. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005884#sec019" target="_blank">Methods</a> for calculation of peak intensity. (<b>D</b>) Venn diagram of RUNX2-dependent transcripts and RUNX2-bound transcripts. (<b>E</b>) Heatmap showing expression changes of the 112 RUNX2 direct targets (fold change >1.5, p<0.05) in SAOS2 cells. Showing is the average of three repeats from RNA-seq. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005884#sec019" target="_blank">Methods</a> for integration of RNA-seq and ChIP-seq. (<b>F</b>) Gene Ontology (GO) terms of genes enriched in the 112 RUNX2 direct targets. (<b>G</b>) Genomic view of RUNX2 and CBFB binding on the MYC locus. The black bars underneath represents identified peaks using the MACS algorithm. (<b>H</b>) I.B. to detect RUNX2, MYC and Tubulin in SAOS2 cells transduced with lentiviruses expressing shLuc, shRUNX2_3 or shRUNX2_4. (<b>I</b>) I.B. to detect CBFB, MYC and Tubulin in SAOS2 cells transduced with lentiviruses expressing shLuc, shCBFB_1 or shCBFB_2. (<b>J</b>) I.B. to detect MYC expression and apoptosis in SAOS2 cells with single or double knockdown of RUNX2 and CBFB.</p

MYC is up-regulated in and required for the survival of OS cells.

<p>(<b>A</b>) I.B. to detect MYC and β-actin in hMSCs and OS cells. (<b>B</b>) Realtime PCR to measure the levels of MYC transcript in hMSCs and OS cells. (<b>C</b>) MYC immunohistochemistry (IHC) and H&E staining of xenografted SAOS2 and Hu09-M112 tumors. Arrows indicate osteoid formation. (<b>D</b>) MYC IHC in OS patient tumors. (<b>E</b>) I.B. of MYC, cleaved caspase 3, and Tubulin in SAOS2 cells. (<b>F</b>) Cumulative cell numbers of SAOS2 cells transduced with lentiviruses expressing shLuc, shMYC_4 or shMYC_5. (<b>G</b>) A model of RUNX2-MYC in p53-independent apoptosis of OS cells.</p

A RUNX2-mediated epigenetic mechanism that regulates MYC expression in OS cells.

<p>(<b>A</b>) A genomic view of H3K4me3 and H3K79me2 ChIP-seq on the MYC locus in SAOS2 cells and hMSCs. The two head-to-head arrows indicate the ChIP amplicon region, which is the RUNX2 binding peak shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005884#pgen.1005884.g005" target="_blank">Fig 5G</a>. (<b>B</b>) ChIP assay to assess the effect of RUNX2 knockdown on the levels of CBFB, H3K4me3, H3K79me2, and Pol II on the MYC promoter in SAOS2 cells. The amplicon region was indicated in (<b>A</b>). (<b>C</b>) RUNX2 and Menin interact in SAOS cells. (<b>D</b>) ChIP assay to assess the effect of RUNX2 knockdown on the levels of Menin, MLL1, and Wdr5 recruitment on the MYC promoter. NoAb, no antibody control. (<b>E</b>) Immunoblotting and (<b>F</b>) flow cytometry showing that MLL1 (upper panels) and Menin (lower panels) knockdown led to the apoptosis of SAOS2 cells.</p

Exogenously expressed MYC partially rescues apoptosis caused by RUNX2 or CBFB knockdown.

<p>(<b>A</b>) Histogram of propidium iodide staining of rescue experiments in SAOS2 cells transduced with empty vector or MYC vector followed by transduction with shLuc, shRUNX2_3 or shRUNX2_4 lentiviruses. (<b>B</b>) Quantitative analyses of sub-G1 phase of the rescue experiments in (<b>A</b>). Error bars are SEM; t-test, **, p<0.01; *, p<0.05. (<b>C</b>) I.B. to detect RUNX2, MYC, cleaved caspase 3, and Tubulin in the rescue experiments in SAOS2 cells transduced with lentiviruses expressing shLuc, shRUNX2_3 or shRUNX2_4. (<b>D</b>) Histogram of propidium iodide staining of rescue experiments in SAOS2 cells transduced with empty vector or MYC vector followed by transduction with lentiviruses expressing shLuc, shCBFB_1 or shCBFB_2. (<b>E</b>) Quantitative analyses of sub-G1 phase of the rescue experiments in (<b>D</b>). Error bars are SEM; t-test, **, p<0.01; *, p<0.05. (<b>F</b>) I.B. to detect CBFB, MYC, cleaved caspase 3, and Tubulin in the rescue experiments in SAOS2 cells transduced with lentiviruses expressing shLuc, shCBFB_1 or shCBFB_2.</p