Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Sep 23:9:258.
doi: 10.1186/1476-4598-9-258.

Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis

Sanjeev K Baniwal  1 , Omar Khalid, Yankel Gabet, Ruchir R Shah, Daniel J Purcell, Deepak Mav, Alice E Kohn-Gabet, Yunfan Shi, Gerhard A Coetzee, Baruch Frenkel
Affiliations

Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis

Sanjeev K Baniwal et al. Mol Cancer. .

Abstract

Background: Prostate cancer (PCa) cells preferentially metastasize to bone at least in part by acquiring osteomimetic properties. Runx2, an osteoblast master transcription factor, is aberrantly expressed in PCa cells, and promotes their metastatic phenotype. The transcriptional programs regulated by Runx2 have been extensively studied during osteoblastogenesis, where it activates or represses _target genes in a context-dependent manner. However, little is known about the gene regulatory networks influenced by Runx2 in PCa cells. We therefore investigated genome wide mRNA expression changes in PCa cells in response to Runx2.

Results: We engineered a C4-2B PCa sub-line called C4-2B/Rx2 dox, in which Doxycycline (Dox) treatment stimulates Runx2 expression from very low to levels observed in other PCa cells. Transcriptome profiling using whole genome expression array followed by in silico analysis indicated that Runx2 upregulated a multitude of genes with prominent cancer associated functions. They included secreted factors (CSF2, SDF-1), proteolytic enzymes (MMP9, CST7), cytoskeleton modulators (SDC2, Twinfilin, SH3PXD2A), intracellular signaling molecules (DUSP1, SPHK1, RASD1) and transcription factors (Sox9, SNAI2, SMAD3) functioning in epithelium to mesenchyme transition (EMT), tissue invasion, as well as homing and attachment to bone. Consistent with the gene expression data, induction of Runx2 in C4-2B cells enhanced their invasiveness. It also promoted cellular quiescence by blocking the G1/S phase transition during cell cycle progression. Furthermore, the cell cycle block was reversed as Runx2 levels declined after Dox withdrawal.

Conclusions: The effects of Runx2 in C4-2B/Rx2 dox cells, as well as similar observations made by employing LNCaP, 22RV1 and PC3 cells, highlight multiple mechanisms by which Runx2 promotes the metastatic phenotype of PCa cells, including tissue invasion, homing to bone and induction of high bone turnover. Runx2 is therefore an attractive _target for the development of novel diagnostic, prognostic and therapeutic approaches to PCa management. _targeting Runx2 may prove more effective than focusing on its individual downstream genes and pathways.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Establishment of C4-2B/Rx2dox sub-line with conditional Runx2 expression. A, Schematic diagram of the pSLIK-based lentiviral vector. Initially developed for expression of shRNAs [39] the pSLIK vector was used in the present study to express Runx2. The Hygromycin resistance marker (Hyg) and the Dox-dependent activator protein rtTA3 are constitutively expressed under the control of the Ubi-c promoter. Upon treatment with Dox, rtTA binds to its tetracycline responsive elements (TRE) and drives expression of the inserted cDNA (Flag-Runx2). The Runx2 block diagram depicts its glutamine/alanine-rich QA domain, the DNA-binding Runt domain, and the proline/serine/threonine-rich PST domain. Arrowhead indicates the position of the R265D and R268D mutations in Runx2-M, which eliminate Runx2's DNA binding function. B, Whole cell extracts, prepared from C4-2B/Rx2dox and C4-2B/Rx2-Mdox cells treated with the indicated concentrations of Dox, were subjected to western blot analysis using anti-Flag antibodies. C, Total RNA was extracted from C4-2B/Rx2dox cells treated with Dox or vehicle, as well as from PC3high cells, and the mRNA levels of Runx2 (and GAPDH as control) were measured by RT-qPCR. D, Whole cell extracts were prepared from C4-2B/Rx2dox cells treated with Dox as indicated and from ROS 17.8/2 osteoblastic cells [41], and subjected to western blot analysis using anti-Runx2 antibodies. The same blot was re-probed with anti-Tubulin antibodies as loading control. E, C4-2B/Rx2dox and C4-2B/Rx2-Mdox cells were transiently transfected with the 6XOSE2-luciferase reporter plasmid and subjected to luciferase assay. Dotted line represents the background luciferase activity with no cell extract. F, C4-2B/Rx2dox cells were treated with Dox and levels of the indicated transcripts were measured by RT-qPCR and corrected for that of GAPDH. In Figure 1, bars represent Mean ± SEM (n = 3) from a representative experiment, which was repeated at least three times with similar results. Abbreviations used: Dox, Doxycycline; Veh, vehicle; BSP, Bone Sialoprotein; MMP9, Matrix Metalloprotein-9; OC, Osteocalcin.
Figure 2
Figure 2
Unsupervised hierarchical clustering of differentially expressed genes. Genes that Runx2 up- or down-regulated by ≥2-fold on either day 1 or day 2 (total: 910 genes) with p < 0.008 were subjected to pearson's centered correlation matrix. Heatmap represents intensity values relative to the median intensity across all 16 samples per probe after background subtraction and normalization.
Figure 3
Figure 3
Runx2-regulated protein expression. C4-2B/Rx2dox cells were treated with Dox or vehicle control, and proteins extracted as whole cell lysate (upper panel) or the supernatant (bottom panel) were subjected to western blot analysis using the indicated antibodies.
Figure 4
Figure 4
Runx2 enhances the invasiveness of C4-2B cells. A) Zymography of supernatants from C4-2B/Rx2dox cells treated with either Dox or vehicle. Negatively-stained bands represent the activity of gelatin-degrading proteases. B) C4-2B/Rx2dox/Luc cells were incubated in the top chambers (inserts) of the Matrigel™ invasion system. Cell passage through inserts with and without Matrigel™ represent invasion and migration, respectively. Identical number of cells was seeded in the indicated inserts for 24 hours and the cells that appeared on the bottom side (outside) of the inserts were solubilized in lysis buffer and subjected to luciferase assay. C) The invasion of cells through the Matrigel™ membrane was assessed by staining with Diff-Quick™ solution.
Figure 5
Figure 5
Runx2 inhibits the G1/S phase transition of the cell cycle. A, RT-qPCR analysis of the indicated genes in C4-2B/Rx2dox cells treated with Dox (filled bars) or vehicle (open bars) for 48 hours. B and C, MTT-based cell proliferation assays of C4-2B/Rx2dox and C4-2B/Rx2-Mdox cells treated with Dox or vehicle as depicted for the indicated time periods. D, Relative apoptosis based on caspase 3 activity in whole cell extracts prepared from C4-2B/Rx2dox and C4-2B/Rx2-Mdox cells after treatment with Dox or vehicle for the indicated time periods. E, FACS-based cell cycle analysis of propidium iodide-stained C4-2B/Rx2dox cells treated with Dox for the indicated time periods. F-I, Schematic description of Dox treatment and its subsequent withdrawal from the cell cultures (F). Samples were harvested at the indicated times (arrows) and subjected to analysis of Runx2 levels by western blotting (G), cell cycle profiling by FACS analysis (H), and cell proliferation by MTT assays (I). Abbreviations: RASD1, RAS, Dexamethasone-induced 1; DUSP, Dual Specificity Phosphatase.
Figure 6
Figure 6
Runx2-regulated cancer-related gene network. The 119 cancer-related genes showing a significant ≥2-fold response to Runx2 on day 1 were subjected to the pathway analysis tool from Ingenuity Systems (IPA™). Direct relationships with Runx2 are shown as thick lines and interactions with its paralogs Runx1 and Runx3 are shown as dashed lines. Red and blue fonts mark up- and down-regulated genes, respectively.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 1997;89:747–754. doi: 10.1016/S0092-8674(00)80257-3. - DOI - PubMed
    1. Cameron ER, Neil JC. The Runx genes: lineage-specific oncogenes and tumor suppressors. Oncogene. 2004;23:4308–4314. doi: 10.1038/sj.onc.1207130. - DOI - PubMed
    1. Lo Coco F, Pisegna S, Diverio D. The AML1 gene: a transcription factor involved in the pathogenesis of myeloid and lymphoid leukemias. Haematologica. 1997;82:364–370. - PubMed
    1. Woolf E, Xiao C, Fainaru O, Lotem J, Rosen D, Negreanu V, Bernstein Y, Goldenberg D, Brenner O, Berke G. et al.Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis. Proc Natl Acad Sci USA. 2003;100:7731–7736. doi: 10.1073/pnas.1232420100. - DOI - PMC - PubMed
    1. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M. et al._targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 1997;89:755–764. doi: 10.1016/S0092-8674(00)80258-5. - DOI - PubMed

Publication types

MeSH terms

  NODES
Association 1
Project 1
twitter 2