Runx2 mediates epigenetic silencing of the bone morphogenetic protein-3B (BMP-3B/GDF10) in lung cancer cells.

Abstract

The Runt-related transcription factor Runx2 is essential for bone development but is also implicated in progression of several cancers of breast, prostate and bone, where it activates cancer-related genes and promotes invasive properties. The transforming growth factor β (TGF-β) family member bone morphogenetic protein-3B (BMP-3B/GDF10) is regarded as a tumor growth inhibitor and a gene silenced in lung cancers; however the regulatory mechanisms leading to its silencing have not been identified.
Here we show that Runx2 is highly expressed in lung cancer cells and downregulates BMP-3B. This inverse relationship between Runx2 and BMP-3B expression is further supported by increased expression of BMP-3B in mesenchymal cells from Runx2 deficient mice. The ectopic expression of Runx2, but not DNA binding mutant Runx2, in normal lung fibroblast cells and lung cancer cells resulted in suppression of BMP-3B levels. The chromatin immunoprecipitation studies identified that the mechanism of Runx2-mediated suppression of BMP-3B is due to the recruitment of Runx2 and histone H3K9-specific methyltransferase Suv39h1 to BMP-3B proximal promoter and a concomitant increase in histone methylation (H3K9) status. The knockdown of Runx2 in H1299 cells resulted in decreased histone H3K9 methylation on BMP-3B promoter and increased BMP-3B expression levels. Furthermore, co-immunoprecipitation studies showed a direct interaction of Runx2 and Suv39h1 proteins. Phenotypically, Runx2 overexpression in H1299 cells increased wound healing response to TGFβ treatment.
Our studies identified BMP-3B as a new Runx2 target gene and revealed a novel function of Runx2 in silencing of BMP-3B in lung cancers. Our results suggest that Runx2 is a potential therapeutic target to block tumor suppressor gene silencing in lung cancer cells.

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RESEARCHOpen Access
Runx2 mediates epigenetic silencing of the bone
morphogenetic protein-3B (BMP-3B/GDF10) in
lung cancer cells
Manish Tandon1†, Karthiga Gokul2†, Syed A Ali2†, Zujian Chen1, Jane Lian2, Gary S Stein2and Jitesh Pratap1*
Abstract
Background: The Runt-related transcription factor Runx2 is essential for bone development but is also implicated in
progression of several cancers of breast, prostate and bone, where it activates cancer-related genes and promotes
invasive properties. The transforming growth factor β (TGF-β) family member bone morphogenetic protein-3B
(BMP-3B/GDF10) is regarded as a tumor growth inhibitor and a gene silenced in lung cancers; however the
regulatory mechanisms leading to its silencing have not been identified.
Results: Here we show that Runx2 is highly expressed in lung cancer cells and downregulates BMP-3B. This inverse
relationship between Runx2 and BMP-3B expression is further supported by increased expression of BMP-3B in
mesenchymal cells from Runx2 deficient mice. The ectopic expression of Runx2, but not DNA binding mutant
Runx2, in normal lung fibroblast cells and lung cancer cells resulted in suppression of BMP-3B levels. The chromatin
immunoprecipitation studies identified that the mechanism of Runx2-mediated suppression of BMP-3B is due to the
recruitment of Runx2 and histone H3K9-specific methyltransferase Suv39h1 to BMP-3B proximal promoter and a
concomitant increase in histone methylation (H3K9) status. The knockdown of Runx2 in H1299 cells resulted in
decreased histone H3K9 methylation on BMP-3B promoter and increased BMP-3B expression levels. Furthermore,
co-immunoprecipitation studies showed a direct interaction of Runx2 and Suv39h1 proteins. Phenotypically, Runx2
overexpression in H1299 cells increased wound healing response to TGFβ treatment.
Conclusions: Our studies identified BMP-3B as a new Runx2 target gene and revealed a novel function of Runx2 in
silencing of BMP-3B in lung cancers. Our results suggest that Runx2 is a potential therapeutic target to block tumor
suppressor gene silencing in lung cancer cells.
Keywords: Lung cancer, Runx2, BMP-3B, Gene silencing
Background
Lung cancer is the leading cause of cancer mortality and
accounts for 30% of all deaths from cancer [1]. Silencing
of tumor suppressor genes by aberrant promoter hyper-
methylation is a key event in lung cancer initiation and
progression. During gene silencing, the chromatin struc-
ture is altered by acetylation, phosphorylation and
methylation of histone tails [2]. These alterations in
chromatin structure affect normal cell functions and are
a crucial trigger for neoplastic development and progres-
sion [3]. However, current understanding of regulatory
mechanisms of silencing of tumor suppressors is limited.
In this study we identified a mechanism by which Runx2
transcription factor contribute to epigenetic silencing of
a tumor growth inhibitor BMP-3B in lung cancer cells.
Runx transcriptionfactors
Runx3) are critical regulators of organogenesis and
cell differentiation regulatory pathways, and mutations
in these genes are associated with several cancers.
Runx2, an essential bone cell differentiation factor
[4,5] is recently implicated in mammary, prostate and
osteosarcoma progression [6-8]. In cancer cells, Runx2
activates cancer-related genes, promotes cells invasive
(Runx1, Runx2and
* Correspondence: jitesh_pratap@rush.edu
†Equal contributors
1Department of Anatomy and Cell Biology, Rush University Medical Center,
Armour Academic Center, 600 S. Paulina St. Suite 507, Chicago, IL 60612, USA
Full list of author information is available at the end of the article
© 2012 Tandon et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Tandon et al. Molecular Cancer 2012, 11:27
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properties [6,8-10], cooperates with oncogenes (e.g., c-myc
in T-cell lymphoma development), and suppresses apop-
totic and growth arrest pathways [11,12]. Runx2 is also a
major target gene of TGFβ /BMP signaling pathway and
the interaction between Runx2 and Smads results in regu-
lation of downstream target genes in osteoblasts [13],
chondrocytes [14] and cancer cells [8].
BMP-3B, a TGFβ family member and closely related
to BMP-3, is highly expressed in lung [15-17], brain
and bone tissues, and induces bone formation [18,19].
Ectopic BMP-3B expression promotes osteoblast differ-
entiation and augments the bone formation induced by
bone morphogenetic protein-2 (BMP-2) in rats [20].
Importantly, the expression of BMP-3B is downregu-
lated in lung cancer patient samples and cancer cells
lines compared to normal lung cells [21-23]. Multiple
mechanisms have been proposed for the downregulation
of BMP-3B levels which include methylation of gene
promoter and repression by transcription factors [21]
however, the transcriptional repressor proteins of BMP-3B
are unknown.
We show that BMP-3B is a novel Runx2 target gene
and find an inverse relationship between Runx2 and
BMP-3B expression levels in normal lung fibroblast and
lung cancer cells. Our studies with Runx2 overexpres-
sion or knockdown in lung cancer cells indicate that
Runx2-mediated downregulation of BMP-3B is via
increasing histone H3K9 methylation status of the
proximal promoter by interacting with methyltransre-
fase Suv39h1.
Results
Calvarial mesenchymal cells of Runx2-deficient mice have
higher expression levels of BMP-3B
To identify novel Runx2 target genes, we performed cDNA
expression analysis on total RNA isolated from calvarial
mesenchymal cells of wild type and functional deficient
Runx2 mice [5]. In addition to the downregulation of
known Runx2 target genes (e.g., matrix metalloproteinases)
in a osteogenesis-related cDNA array [24], we found that
the expression levels of BMP-3B gene was induced in
Runx2 deficient cells compared to wild type cells
(Figure 1a). The induction of BMP-3B expression in
Runx2 deficient calvarial mesenchymal cells was vali-
dated by qRT-PCR analysis (Figure 1b). To further
confirm Runx2-mediated downregulation of BMP-3B
levels, we re-expressed Runx2 via adenoviral delivery
in Runx2 deficient primary calvarial cells and measured
BMP-3B levels by qRT-PCR analysis (Figure 1c). Our
results show a dose-dependent repression of BMP-3B
mRNA levels by Runx2 in primary osteoblastic cells.
These results suggested that BMP-3B is a novel Runx2
responsive gene.
An inverse relationship between Runx2 and BMP-3B
expression levels in lung cancer cells
A tumor growth inhibitory function was proposed for
BMP-3B in lung cancers and BMP-3B is downregulated
in most of the lung cancers [22,23,25]. In context of
Runx2-mediated BMP-3B suppression in mesenchymal
cellsandtounderstand
mechanisms of BMP-3B silencing in lung cancers, we
hypothesized that Runx2 downregulates BMP-3B expres-
sion in lung cancer. To understand the role of Runx2 in
BMP-3B transcriptional regulation in lung cancer cells,
we first examined Runx2 and BMP-3B mRNA levels in
normal lung fibroblasts of mesenchymal origin (WI-38
and IMR-90), atypical carcinoid (H720) and meta-
static non-small cell lung carcinoma (H1299) cells by
qRT-PCR analysis. Our results showed that Runx2
expression is increased in metastatic lung cancer cells
(H1299) compared to normal lung fibroblast cells. In
contrast to the Runx2 expression levels, BMP-3B
mRNA was detectable but lower in lung cancer cells
compared to normal lung fibroblast cells (Figure 1d
and e). The Western blot analysis for Runx2 protein
levels further validated increased Runx2 levels in lung
cancer cells compared to normal lung fibroblast cells
(Figure 1f). A punctate nuclear staining of Runx2
was observed in WI-38 and H1299 cells as examined
by immunofluorescence (Figure 1g). Taken together,
these studies revealed that the inverse relationship
between Runx2 and BMP-3B levels observed in cal-
varial mesenchymal cells also holds true for normal
lung fibroblasts and lung cancer cells.
theupstream regulatory
Runx2 overexpression suppresses BMP-3B in lung cancer
cells
To investigate whether Runx2 suppresses BMP-3B levels
in lung cancer cells similar to observed in primary cal-
varial cells, we stably overexpressed wild type Runx2
(WT) and Runx2 DNA binding domain mutant (DBD)
in normal lung fibroblast cells (WI-38 and IMR-90) by
lentiviral-mediated gene delivery. Expression levels of
wild type and mutant Runx2 protein in these cell types
were confirmed by qRT-PCR and western blot analysis
(data not shown). Our results showed that stable expres-
sion of wild type Runx2 in normal lung cells resulted in
more than 2-fold decrease in BMP-3B levels compared
to empty vector control cells (Figure 2a and b). Ectopic
expression of DBD mutant of Runx2 failed to downregu-
late BMP-3B levels in normal lung or lung cancer cells.
These results suggested that the Runx2 DNA binding
activity is required for BMP-3B regulation. In complemen-
tary studies, Runx2 knockdown resulted in increased BMP-
3B levels in normal bronchial NL-20 cells (10- fold increase,
Figure 2c right panel) and H1299 cells (2-fold increase,
Figure 2d right panel) compared to empty vector controls
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as shown by qRT-PCR analysis. The decrease in Runx2
levels in Runx2 knockdown cells was confirmed by qRT-
PCR and western blot analysis (Figure 2c and d). Collect-
ively, these results indicate that Runx2 downregulates BMP-
3B levels in normal lung fibroblast and lung cancer cells.
Runx2 recruitment on the BMP-3B gene promoter and
interaction with Suv39h1 promotes BMP-3B silencing
To further investigate the mechanism of Runx2-mediated
downregulation of the BMP-3B expression in lung cancer
cells, we performed chromatin immunoprecipitation ana-
lysis in H1299 cells expressing either wild type Runx2 or
shRunx2 (Figure 3). Our results showed 3-fold increased
Runx2 binding on the BMP-3B proximal promoter
(-1.0kb) in H1299-WT-Runx2 cells, that was abrogated in
H1299-shRunx2 cells. We next examined the methylation
status of the BMP-3B proximal promoter as methylation
of lysine 9 of histone H3 (H3K9) allows the binding of het-
erochromatin protein- 1 (HP1) to silence gene expression
[26,27]. Our results show increased (3-fold) H3K9 levels of
proximal promoter region of BMP-3B in H1299-Runx2
cells compared to H1299-shRunx2 cells or antibody con-
trols (Figure 3a). We next examined the recruitment of
Suv39h1 protein, a histone H3 lysine 9 specific methyltrans-
ferase, on BMP-3B proximal promoter. A twofold increase
in recruitment of Suv39h1 was observed in H1299-Runx2
cells compared to H1299-shRunx2 lung cancer cells
(Figure 3a). These findings indicated the possibility of
physical interaction of Runx2 and Suv39h1 proteins in
lung cancer cells. We performed co-immunoprecipitaion
assays with Runx2 and Suv39h1 antibodies and a direct
interaction of Runx2 with Suv39h1 proteins was detected in
H1229 cells (Figure 3b). Taken together, these results show
that the recruitment of Runx2 and Suv39h1 on the BMP-3B
Figure 1 Runx2 deficient mice have higher levels of BMP-3B. (a) Total RNA from mesenchymal cells of calvarial tissue from wild type and
Runx2-/-(17.5 dpc) was hybridized with osteogenesis-related cDNA array. Signal of BMP-3B on blots is indicated by an arrow. Control genes
(GAPDH, cyclophilin A) are shown in lower panel. (b) BMP-3B mRNA levels (normalized to GAPDH) in calvarial mesenchymal cells from wild type
(WT) and Runx2-/-animals as detected by qRT-PCR analysis. (c) Primary calvarial cells from Runx2 deficient animals were transduced with empty
vector (EV) or Runx2 expressing adenovirus with increasing multiplicity of infection (MOI 10 and 20) for 48 hr. Total RNA isolated from the infected
cells was utilized to examine BMP-3B mRNA as detected by qRT-PCR analysis. (d) Runx2 mRNA expression levels were examined by qRT-PCR and
the gene expression levels were normalized to 28S internal control. The gene expression levels were calibrated to IMR-90 cells. (e) BMP-3B mRNA
expression levels were examined by qRT-PCR and the gene expression levels were normalized to 28S internal control. The BMP-3B gene
expression levels were calibrated to Runx2 levels in IMR-90 cells. (f) Runx2 protein levels were examined in nuclear extracts of normal lung
fibroblast cells (IMR-90 and WI-38) and lung cancer cell lines (H720 and H1299) by immunoblotting with Runx2 monoclonal antibody. Runx2
expression levels were normalized to internal control LaminA/C protein. (g) Runx2 intracellular localization was determined by
immunofluorescence of endogenous Runx2 protein in WI-38 and H1299 cells. The punctate (Alexa 488, green) signal shows Runx2 staining while
nuclei are revealed by 4, 6-diamidino-2-phenylindole (DAPI, blue) staining.
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proximal promoter sequences resulted in increased H3K9
methylation status and consequently downregulation of
BMP-3B expression in lung cancer cells.
Runx2 increases wound-healing response of lung cancer
cells
To examine the phenotypic effects of Runx2 overexpression
in lung cancer cells, we assessed proliferation and migration
potential of H1299-Runx2 cells or H1299- empty vector
cells. Increased Runx2 levels in H1299-Runx2 cells and a
corresponding decrease in BMP-3B mRNA expression were
confirmed by western blot and qRT-PCR analysis respect-
ively (Figure 4a). A 40% decline in cell proliferation was
observed in Runx2 overexpressing H1299 cells compared to
empty vector control cells in absence or presence of TGFβ
treatment as examined by cell growth assay (data not
shown) and MTT assays (Figure 4b). However, in response
to TGF-β treatment the Runx2 overexpression in H1299
cells resulted in a significant (p <0.05) increase in wound
healing response compared to the empty vector control for
6-48h as shown by wound healing assay (Figure 4c). The
H1299 EV or WT-Runx2 cells did not show any differences
in KI-67 (cell proliferation marker) immunoreactivity
around wound area (data not shown). These results suggest
that Runx2 promotes migratory potential of lung cancer
cells by modulating TGF-β/BMP-3B signaling axis.
Discussion
Our studies identify BMP-3B as a Runx2 target gene and
show that Runx2 promotes epigenetic silencing of BMP-3B
Figure 2 Runx2 suppresses BMP-3B in lung cancer cells. (a and b) Expression levels of BMP-3B in normal lung fibroblast cells (a: WI-38, b:
IMR-90) transduced with wild type Runx2 (WT), functional deficient mutant (DBD: DNA binding domain mutant,) and empty vector (EV) control
lentiviral particles are shown as detected by qRT-PCR analysis. (c and d) The Runx2 protein was suppressed in normal bronchial NL-20 epithelial
cells and H1299 lung cancer cells by lentiviral vector-mediated RNAi. The suppression of Runx2 at mRNA and protein expression levels is shown
for NL-20 cells (C top left and lower left panels). The mRNA levels of BMP-3B in Runx2 suppressed NL-20 and control cells are shown as examined
by qRT-PCR (C top right panel). The suppression of Runx2 at mRNA and protein expression levels is shown for H1299 cells (D top left and lower
left panels). The mRNA levels of BMP-3B in Runx2 suppressed H1299 and control cells as examined by qRT-PCR (D top right panel).
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in lung cancer cells by promoting histone H3K9 methyla-
tion status of the proximal regulatory regions. The Runx2
interaction with Suv39h1 methyltransferase and binding to
the BMP-3B promoter results in downregulation of the
BMP-3B expression levels. Furthermore, ectopic expression
of Runx2 enhances the migration potential of lung cancer
cells in response to the TGFβ signaling.
We find that mesenchymal cells from Runx2-deficient
animals express high levels of BMP-3B compared to wild
type cells. In contrast to high levels of BMP-3B, low
baseline levels of BMP2 are reported in Runx2 deficient
cells that can be up-regulated by ectopic expression of
Runx2 [28]. Interestingly, a BMP2 orthologous signaling
antagonizing function for BMP3/3B has been proposed
during embryonic development of xenopus [29]. In
addition to directly regulating expression levels of BMP
family members as shown by these studies, Runx2-Smad
complex has been shown to regulate expression of genes
related to osteogenic [10,30,31] and cancer [32] properties
in response to TGFβ/BMP signaling. The consequences of
direct regulation of BMP-3B by Runx2 on downstream mo-
lecular events of TGFβ/BMP pathway still need to be deter-
mined. A recent report shows that the migration of lung
cancer cells is associated with the upregulation of Runx2
and Snail expression in response to BMP-2 treatment [33].
Our results show that Runx2 downregulates BMP-3B and
increases migration potential of lung cancer cells in re-
sponse to TGFβ treatment. These studies suggest that
cross-talk between Runx2 and TGFβ/BMP signaling is dif-
ferential and could be context-dependent.
Figure 3 Runx2 recruitment on BMP-3B gene promoter and interaction with Suv39h1. (a) Lung cancer cells (H-1299) stably expressing wild
type Runx2 (left panel) or shRNA-Runx2 (right panel) were subjected to chromatin immunoprecipitation assay with Runx2, Suv39h1, H3K9 or
control IgG-G (goat) and IgG-R (rabbit) antibodies. Immunoprecipitated DNA samples were further amplified by real-time qPCR with primer pairs
for proximal promoter (-1.0kb) of BMP-3B gene. A schematic diagram above indicates Runx binding motifs (solid bars) on BMP-3B promoter. The
BMP-3B ChIP primer pairs are indicated by arrows in the schematic diagram. (b) Co-immunoprecipitation assay shows Runx2 interaction with
Suv39h1 in H-1299 lung cancer cells. Whole cell extracts from lung cancer H-1299 cells were incubated with Runx2, Suv39h1 or control IgG
antibodies. Western blots showing Runx2 and Suv39h1 proteins as detected by αRunx2 and αSuv39h1 antibodies.
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