Cell Reports
Article
TET1 Suppresses Cancer Invasion by Activating
the Tissue Inhibitors of Metalloproteinases
Chih-Hung Hsu,
1,11,13Kai-Lin Peng,
1,2,11Ming-Lun Kang,
1,12Yi-Ren Chen,
3,12Yu-Chih Yang,
3Chin-Hsien Tsai,
3,4Chi-Shuen Chu,
1,5Yung-Ming Jeng,
6Yen-Ting Chen,
1,5Feng-Mao Lin,
7Hsien-Da Huang,
7Yun-Yuh Lu,
1Yu-Ching Teng,
1,2Shinn-Tsuen Lin,
8Ruo-Kai Lin,
1,14Fan-Mei Tang,
9Sung-Bau Lee,
1,15Huan Ming Hsu,
10Jyh-Cherng Yu,
10,*
Pei-Wen Hsiao,
3,*
and Li-Jung Juan
1,2,5,*
1Genomics Research Center, Academia Sinica, Taipei 115, Taiwan, ROC
2Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan, ROC 3Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC
4Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan, ROC
5Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ROC 6Graduate Institute of Pathology, National Taiwan University, Taipei 100, Taiwan, ROC
7Department of Biological Science and Technology, Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu 300, Taiwan, ROC
8R&D department, GeneTex Asia Ltd, Hsinchu 300, Taiwan, ROC
9Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC
10Division of General Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan, ROC 11These authors contributed equally to this work
12These authors contributed equally to this work
13Present address: Division of Newborn Medicine, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115, USA 14Present address: Graduate Institute of Pharmacology, Taipei Medical University, Taipei 110, Taiwan, ROC
15Present address: Biotech Research and Innovation Centre (BRIC), University of Copenhagen, DK2200 Copenhagen, Denmark *Correspondence:[email protected](J.-C.Y.),[email protected](P.-W.H.),[email protected](L.-J.J.)
http://dx.doi.org/10.1016/j.celrep.2012.08.030
SUMMARY
Tumor suppressor gene silencing through cytosine
methylation contributes to cancer formation. Whether
DNA demethylation enzymes counteract this
onco-genic effect is unknown. Here, we show that TET1,
a dioxygenase involved in cytosine demethylation, is
downregulated in prostate and breast cancer tissues.
TET1 depletion facilitates cell invasion, tumor growth,
and cancer metastasis in prostate xenograft models
and correlates with poor survival rates in breast
cancer patients. Consistently, enforced expression
of TET1 reduces cell invasion and breast xenograft
tumor formation. Mechanistically, TET1 suppresses
cell invasion through its dioxygenase and DNA
binding activities. Furthermore, TET1 maintains the
expression of tissue inhibitors of metalloproteinase
(TIMP) family proteins 2 and 3 by inhibiting their
DNA methylation. Concurrent low expression of
TET1 and TIMP2 or TIMP3 correlates with advanced
node status in clinical samples. Together, these
re-sults illustrate a mechanism by which TET1
sup-presses tumor development and invasion partly
through downregulation of critical gene methylation.
INTRODUCTION
Tumor growth and cancer formation is controlled by both genetic
and epigenetic events (Dawson and Kouzarides, 2012;
You and
Jones, 2012). Cancer epigenetic events include aberrant DNA
methylation such as localized CpG island hypermethylation
that leads to inactivation of specific tumor-suppressor genes
(Kulis and Esteller, 2010;
Taberlay and Jones, 2011). While
DNA methyltransferases have been demonstrated to be
onco-genic by silencing tumor-suppressor genes via cytosine
methyl-ation, whether enzymes with opposing function protect cells
from becoming malignant remains elusive.
The ten eleven translocation (TET) family of proteins, TET1,
TET2, and TET3, were discovered on the basis of TET1 fusion to
the mixed lineage leukemia gene in acute myeloid leukemia
(Lors-bach et al., 2003;
Ono et al., 2002). In 2009, TET1 was found to be
a dioxygenase that converts 5-methylcytosine (5mC) to
5-hydrox-ymethylcytosine (5hmC) (Tahiliani et al., 2009). The same activity
of TET1 and its role in mouse embryonic stem cells was identified
by another independent group (Ito et al., 2010). Moreover, TET
proteins were demonstrated to be capable of further converting
5hmC to 5-formylcytosine and 5-carboxylcytosine, which can
be excised by thymine DNA glycosylase (He et al., 2011;
Ito
et al., 2010;
Ito et al., 2011;
Tahiliani et al., 2009;
Wu and Zhang,
2010). Functions of TET proteins are believed to rely on the
CXXC domain for DNA binding in TET1 and on the
double-stranded
b helix domain for the 2-oxoglutarate- and
Fe(II)-depen-dent oxygenase activity (Wu and Zhang, 2011a;
Xu et al., 2011b).
Importantly, not only are TET proteins potentially involved in the
active DNA demethylation process, they have also been shown
to prevent DNA methylation by physically binding to DNA (Wu
and Zhang, 2011a). Whether TET proteins play any role in cancer
formation remains an intriguing question. It has been revealed that
the catalytic TET2 mutations with decreased 5hmC are associated
with myeloid cancers (Ko et al., 2010). Loss of TET1 and 5hmC in
solid tumors is recently reported as well (Haffner et al., 2011;
Jin
et al., 2011;
Kudo et al., 2012;
Yang et al., 2012). However, to
date, the underlying mechanism and target genes involved in the
TET family of proteins’ function in cancer are unknown.
Cell invasion is one of the pivotal steps in both primary tumor
growth and metastasis initiation (Deryugina and Quigley, 2006;
Friedl and Alexander, 2011;
Hojilla et al., 2008;
Hua et al.,
2011). The matrix metalloproteinase (MMP) family of proteins
and their endogenous regulators, tissue inhibitors of
metallopro-teinases (TIMPs), are key players in the modulation of cell
invasion and epithelial-mesenchymal transition (Bourboulia and
Stetler-Stevenson, 2010;
Kessenbrock et al., 2010;
Murphy,
2011). For example, MMP2 and MMP9 stimulate cell invasion
by inducing basement membrane damage and focal degradation
of type IV collagen and laminin (Zeng et al., 1999). Although it is
not completely understood, one of the major functions of TIMP
proteins is to inhibit the activities of MMPs by binding to the active
site of MMPs or sequestering the pro-MMP zymogens (Clark
et al., 2008;
Murphy, 2011). Interestingly, the expression of
some of the TIMP genes is known to be regulated by DNA
meth-ylation (Chernov et al., 2009).
In the current study, we demonstrate the suppressive role
of TET1 in both prostate and breast cancer invasion by using
cell-based assays, xenograft models, and human clinical
tis-sue samples. Furthermore, we show that the MMP inhibitors
TIMP2 and TIMP3 are pivotal TET1 downstream effectors
responsible for TET1-mediated invasion suppression.
RESULTS AND DISCUSSION
TET1 Expression Is Downregulated in Prostate Cancer
Given the established role of DNA methylation in cancer, we
investigated whether TET1, a protein recently shown to play
A
B
Figure 1. TET1 Is Decreased in Human Prostate Cancer Tissues and Implanted Prostate Cancer Cells Metastasized from Mouse Prostate to Lung
(A) IHC detection of TET1 protein in normal and cancerous human prostate tissues. Shown in right are three representative examples of normal adjacent tissues and tumor sections. Original magnification,3200 (normal), 3400 (cancer). (B) TET1 expression inversely correlates with prostate cancer progression. Relative TET1 mRNA and protein levels in human prostate cancer 22Rv1 cells from mouse prostate at week 2 (2W) or 15 (15W), or from lung at week 15 (lung) after implantation of 22Rv1 into the prostate of nude mice are shown as mean ± SD from triplicate experiments for RT-PCR. p values were measured by Student’s t test. *p < 0.05; **p < 0.01.
a role in regulating DNA demethylation,
is involved in tumor development. In
an examination of TET1 expression in
different human tissues, we found that
TET1 mRNA level was enriched in
pros-tate, breast, and ovary tissues as
com-pared to lung, liver, and colon tissues (data not shown),
indicating that TET1 might have important functions in these
endocrine-related tissues. We then hypothesized that, if TET1
serves as a tumor suppressor, loss of TET1 might play a role in
the development of tumors derived from these TET1-rich
tissues. To test this hypothesis, we first investigated prostate
cancer. Immunohistochemical staining demonstrated that 33%
of 153 prostate cancer patients showed reduced TET1
expres-sion in cancerous cells as compared to non-neoplastic glands,
while 67% had equal TET1 levels (Figure 1A). Note that the
samples were diagnosed as prostate cancer cells via H&E
staining and stained with rabbit IgG as a negative control (data
not shown). Furthermore, we found that TET1 downregulation
was likely involved in cancer metastasis, as demonstrated in
the following experiments. To evaluate the role of specific genes
in cancer metastasis, we previously developed a mouse system
(Tsai et al., 2009) in which highly invasive and
puromycin-resistant human prostate cancer 22Rv1 cells tagged with a
luciferase reporter gene were orthotopically implanted into the
prostate of nude mice. At different times after implantation, cells
from prostate or lung tissue were evaluated for the expression of
luciferase, followed by isolation with puromycin selection. In this
way, the metastasis of implanted cells can be analyzed. Using
this animal model, we demonstrated that both the mRNA and
protein levels of TET1 were dramatically reduced during the
metastasis of transplanted 22Rv1 cells from prostate to lung
(Figure 1B), indicating that TET1 expression is negatively
associ-ated with the progression of prostate cancer metastasis.
TET1 Suppresses Prostate Cancer Invasion and
Metastasis
To evaluate whether TET1 indeed plays a role in the metastasis
process, we generated a TET1 knockdown shRNA (designated
KD1, used throughout the study) and confirmed its efficiency in
depleting TET1 mRNA (Figure S1A) and protein (Figure S1B)
from cells. The TET1 shRNA also increased and decreased the
levels of 5mC and 5hmC, respectively (Figure S1C). Consistent
with a potential role of TET1 in suppressing cancer metastasis,
knocking down TET1 facilitated migration and invasion of both
low (LNCaP) and high (22Rv1) invasive prostate cancer cells in
an in vitro cell-based assay (Figures 2A, 2B and
S2).
Interest-ingly, we noted that TET1 depletion slightly increased prostate
cancer cell proliferation (Figure S3). Nevertheless, the fold
increase of migration was more than the fold increase of
prolifer-ation in both LNCaP and 22Rv1 cells depleted of TET1 (compare
-40 0 40 80 1 0 2 0 3 0 4 0 5 0 SC TET1-KD 0 200 400 600 800 1000 Tum or w ei gh t ( m g ) P=0.0001
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-3 0 3 6 9Rel
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10 20 30 40 50 Time (hr)B
Invasion- LNCaP
SC TET1-KD 0 5 10 15 20 10 20 30 40 50 Time (hr)Invasion- 22Rv1
SC TET1-KD Time (hr) SC TET1-KDMigration- 22Rv1
10 20 30 40 50Rel
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-10 0 10 20 30 10 20 30 40 50 Time (hr)Migration- LNCaP
SC TET1-KD *** *****
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W1 W3 W5 W7 W9 W11 SC TET1-KD Color Bar Min=7e+05 Max=1e+09Rel
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TET1-KD SC 1000 400 200 SC TET1-KD Tum or w ei g h t (m g)***
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SC TET1-KDE
Color Bar Min=5e+05 Max=5e+06 SC TET1-KD 105 106 107 108 109 1010 L u c if er ase act ivit y (photons /s e c ) p=0.04 Liver Liver GI GI Kidney Kidney Spleen Spleen Pancreas Pancreas Diaphragm Diaphragm SC TET1-KDF
SC TET1-KD L u cife ra se a c tiv ity log 10 (phot ons /s ec )Figure 2. TET1 Depletion Stimulates Prostate Cancer Invasion and Metastasis In Vitro and In Vivo
(A and B) TET1 knockdown promotes migration (A) and invasion (B) of prostate cancer cells LNCaP and 22Rv1. The migrating or invading cells with scramble shRNA (SC) or TET1 shRNA (TET1-KD) were analyzed by Roche xCELLigence, and the data are shown as cell index curves with mean± SD from triplicate experiments.
(C–E) TET1 knockdown facilitates the tumorigenesis (C and D) and metastasis (E) of LNCaP-derived prostate cancer xenografts in nude mice.
(C) The luciferase images of the cancer cells in representative mice taken at the indicated time. A total of eight and seven mice were analyzed in the control and TET1-KD groups, respectively.
(D) Left: photograph of dissected tumors from mice taken at week 12 after implantation. Right: relative tumor weight.
(E) Left: bioluminescence images of the host lungs from mice taken at week 12 after implantation. Right: microscopic examination with H&E staining for the verification of in situ pulmonary metastasis (arrow).
(F) TET1 depletion facilitates metastasis of 22Rv1-derived prostate cancer xenografts in nude mice. Left: ex vivo image of the peritoneal organs isolated from each group of mice at endpoint. Right: quantification of the bioluminescent image on the left.
Figure 2A with
Figure S3). Therefore, it is unlikely that the
observed increase in migration and invasion of TET1-deficient
cells was simply due to the enhanced cell proliferation.
To substantiate the in vitro observations in vivo, we analyzed
whether depletion of TET1 promotes prostate cancer cell
metas-tasis in mice. To this end, control and TET1-depleted prostate
cancer cells tagged with luciferase were injected into the
pros-tate of nude mice, followed by analysis of proliferation and
metastasis of the injected cells at week 12 after implantation.
The results demonstrated that TET1-depleted LNCaP (Figures
2C and 2D) or 22Rv1 cells (Figures S4A and S4B) formed larger
tumors. Xenograft experiments further demonstrated that
elimi-nation of TET1 facilitated metastasis of transplanted 22Rv1
(Figure S4C) or LNCaP (Figures 2E,
S4D, and S4E) cells from
prostate to lung in vivo, as evidenced by the metastasis
micro-scopically observed in lung tissue. Increased metastasis of
TET1-depleted 22Rv1 cells to organs other than the lung was
also observed (Figure 2F). Collectively, the results above indicate
that TET1 most likely inhibits prostate cancer metastasis and the
underlying mechanism may involve TET1-mediated
downregula-tion of cell migradownregula-tion and invasion.
TET1 Inhibits Breast Cancer Cell Invasion and Xenograft
Tumor Growth
Subsequently, we asked whether the role of TET1 in suppressing
prostate cancer metastasis can be extended to breast cancer.
To this end, we eliminated TET1 from normal (M10) and
cancerous (MDA-MB-231) breast cells (Figures S1A and S1C)
and analyzed its effect on cell invasion. The results shown in
Fig-ure 3A and
Figure S5A demonstrated that TET1 depletion by
TET1 shRNA KD1 increased the cell-invasion capacity. Using
another independent TET1 shRNA, KD2, we observed a similar
effect in M10 cells (Figures S5B and S5C). Consistently, ectopic
expression of TET1 reduced cell invasion (Figure 3B). Note that
TET1 depletion decreased the proliferation of both M10 and
MDA-MB-231 cells (Figure S5D), ruling out the possibility that
the increased invasion by TET1 shRNA was due to enhanced
cell proliferation. To evaluate whether the invasion suppression
capacity of TET1 requires its enzymatic or CpG binding activity,
we generated a TET1 mutant with amino acid substitutions in the
catalytic domain (CD mut) or a deletion in the CpG binding
domain CXXC (dCXXC mut) and expressed the mutants and
wild-type TET1, respectively (Figure S6A). We then used a dot
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Time (hr) Vector TET1 0 20 40 Vector TET1 10 20 30 40 50Invasion- M10
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**
**
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Vector
TET1
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Tum
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p=0.022 20 60 100 140 180 Tum o r w e igh t (m g)Figure 3. TET1 Inhibits Breast Cancer Cell Invasion and Tumor Formation
(A) TET1 knockdown stimulates cell invasion. The invading M10 (left) or MDA-MB-231 (right) cells with or without TET1 shRNA were analyzed by Roche xCELLigence, and the data are shown as cell index curves with mean± SD from triplicate experiments.
(B) TET1 overexpression represses cell invasion. The invading M10 or MDA-MB-231 cells with or without expression of Flag-tagged TET1 were analyzed for invasion ability as in (A).
(C) TET1 suppression of cell invasion requires intact catalytic and CXXC domains. MDA-MB-231 cells expressing TET1 shRNA were transfected with vector alone, WT TET1 (TET1), catalytic mutant (CD mut), or CXXC-deleted mutant (dCXXC), followed by cell invasion analysis at 24 hr by Roche xCELLigence. (D) Inducible expression of TET1 inhibits breast xenograft tumor formation. Left: photograph of mammary tumors from 4T1-vector and 4T1-TET1 cells, dissected at week 3 after implantation. Right: quantification of the bioluminescent image on the left.
Data from (A) to (C) are shown as mean± SD. p values were measured by repeated-measure ANOVA in (A) and (B), and by Student’s t test in (C) and (D). **p < 0.01; ***p < 0.001.
blot assay to confirm that the CD mut did lose the ability to erase
the cellular 5mC signal, while the dCXXC mut still retained the
ability (Figure S6B). The phenotype of the dCXXC mut is
consis-tent with that observed in a previous study (Frauer et al., 2011).
Results shown in
Figure 3C demonstrated that re-expression
of wild-type (WT) TET1, but not the catalytic mutant, suppressed
cell invasion induced by TET1 depletion. In addition, the dCXXC
mut failed to repress cell invasion as efficiently as WT TET1
(Fig-ure 3C). These results not only support the specificity of the TET1
shRNA used but also demonstrate that both the dioxygenase
and CpG binding activities are pivotal for TET1 function in
invasion suppression. Indeed, TET1 is known to exert its effect
through both enzymatic-activity-dependent and -independent
manners. It has been reported that, in addition to actively
deme-thylating cytosines, TET1 is able to prevent DNA methylation by
physically occupying the unmethylated cytosines (Wu et al.,
2011;
Xu et al., 2011b). The importance of the catalytic and
DNA binding domains in TET1 function was recapitulated in the
following experiment. Consistent with the notion that TET1 is
most likely a suppressor of breast cancer invasion, consecutive
selection in an in vitro invasion assay by six rounds greatly
reduced TET1 mRNA levels in 468-6 cells, the highly invasive
fraction of MDA-MB-468 cells (Figure S7A). As expected,
re-expression of WT TET1, but not the enzymatic or CXXC mutant,
inhibited the cell-invasion capacity of 468-6 cells (Figure S7B).
This is consistent with the observation that enzymatic activity
and CpG binding capacity are required for TET1-mediated
inva-sion suppresinva-sion.
To further understand whether the invasion-suppression
func-tion of TET1 in breast cancer cells can be observed in vivo, we
analyzed tumor development from cells with or without stably
inducible expression of TET1. As shown in
Figures 3D and
S8,
mouse mammary carcinoma 4T1 cells with human TET1
expres-sion generated significantly smaller tumors, further confirming
the tumor-suppressor role of TET1 in breast cancer.
TET1 Downregulation in Breast Cancer Correlates with
Poor Survival Outcomes in Patients
To determine whether the observations above can be verified in
human breast cancer tissues, we analyzed TET1 mRNA levels in
140 breast cancer tissue samples and their normal control
tissues by quantitative real-time RT-PCR, using actin as an
internal control (Figure S9). Of the 140 pairs of sample tissue,
95 (68%) exhibited lower levels of TET1 mRNA in cancer tissues
as compared with their normal-tissue control counterparts
(Fig-ure 4A). Importantly, the lowest tertile of breast cancer patients
(n = 49) found to have low expression levels of TET1 had larger
tumors with advanced stage (Figure 4B) and poor survival
(Fig-ure 4C) as compared with the highest tertile of patients (n = 49)
with high TET1 expression levels. Similar survival correlation
was observed in another independent cohort of 96 breast cancer
patients. Moreover, in this second cohort, up to 81% and 79% of
patients showed decreased TET1 expression in cancer tissues
when actin and GAPDH, respectively, were used as the internal
controls (Figure S10). Together, these results demonstrated that
the TET1 expression level was inversely correlated with breast
cancer cell invasion and tumor development, indicating that
TET1 might play an important role in suppressing breast cancer
progression.
TET1 Promotes the Expression and Function of TIMP2
and TIMP3
Our data indicate that TET1 very likely suppresses the invasion of
both prostate and breast cancers. To understand how TET1 is
A
B
C
Figure 4. TET1 Downregulation Correlates with Advanced Stage and Poor Survival in Breast Cancer Patients
(A) TET1 is downregulated in the majority of the breast cancer tissues. The mRNA level of TET1 in human cancerous breast tissue specimens or adjacent normal tissues was analyzed and normalized to actin. In 140 sample pairs, 95 (68%) have decreased levels of TET1 mRNA in cancer tissues (green), 40 (29%) show equal TET1 mRNA levels (blue), and 5 (3%) exhibit higher levels of
TET1 mRNA in cancerous tissues (red).
(B) TET1 downregulation positively correlates with larger tumor and advanced stage of breast cancer. Patients were grouped into tertiles based on TET1 expression levels. 49 patients with the lowest or highest expression levels of TET1 in the cancer tis-sues from 144 breast cancer patients, overlapping with 140 patients in (A), were analyzed in terms of the relationship between TET1 expression and clinical parameters including age, tumor size, cancer stage, tumor status, and node status. Stage was deter-mined according to the AJCC system. p values were measured according to methods indicated at the bottom of the panel.**p < 0.01; ***p < 0.001. (C) TET1 expression inversely correlates with breast cancer patient survival. The same sets of breast cancer patients as in (B) were analyzed. The survival rates of breast cancer patients were estimated by Kaplan-Meier analysis. Log-rank test was used to compare the survival rates between the upper and lower tertile of patients on the basis of TET1 expression level.
involved in this process, we performed microarray studies.
Comparison of gene expression between control and
TET1-depleted M10 cells indicated that 406 genes were differentially
expressed (90 upregulated and 316 downregulated)
(Fig-ure S11A). We confirmed the fidelity of the microarray results
by analyzing representative genes with real time RT-qPCR
(Figures S11B–S11D). Given that TET1 suppresses invasion
and metastasis, particular attention was paid to the
TET1-regu-lated genes implicated in these processes. Among the 406
potential targets, the TIMP family genes TIMP2 and TIMP3 are
of great interest, not only because of their ability to modulate
MMPs (Bourboulia and Stetler-Stevenson, 2010;
Kessenbrock
et al., 2010;
Murphy, 2011) but also because their expression
is known to be regulated by DNA methylation (Chernov et al.,
2009). The TIMP family comprises the genes TIMP1, TIMP2,
TIMP3, and TIMP4. We found that TET1 depletion in two types
of cancerous prostate cells (LNCaP and 22Rv1) and in normal
and cancerous breast cells (M10 and MDA-MB-231,
respec-tively) resulted in downregulation of TIMP2 and TIMP3
expres-sion (Figure 5A), indicating that TET1 is a positive regulator of
these two genes. mRNA levels of TIMP2 and TIMP3 were also
found to be diminished in other cells, including human breast
cancer cells MCF7 and MDA-MB-468, when TET1 was depleted
(Figure S11E). In contrast, the TIMP1 mRNA level did not seem to
be altered by TET1 in a consistent manner: it was slightly
upregu-lated in M10 and only minimally downreguupregu-lated in MDA-MB-231
when TET1 was knocked down (Figure 5A). TIMP4 was
unde-tectable in both M10 and MDA-MB-231 cells.
Subsequently, we investigated whether the concurrent
expression of TET1 and TIMP2 or TIMP3 observed in cell lines
can be recapitulated in vivo. As shown in
Figure S12, similar to
that of TET1, the TIMP3 mRNA level was also downregulated
in implanted 22Rv1 cells metastasized from mouse prostate to
lung. Furthermore, breast cancer patients with lower levels of
TET1 expression had significantly lower levels of TIMP2 and
TIMP3 expression (
Figure 5B). Analysis of the same set of breast
cancer patients with high expression of TET1 and TIMP2 or
TIMP3 or low expression of TET1 and TIMP2 or TIMP3 indicated
that concurrent low expression of TET1 and TIMP2 or TIMP3 is
correlated with advanced node status (Figure 5C). These studies
not only strongly support the hypothesis that TET1 is an
upstream activator for the expression of TIMP2 and TIMP3 but
also provide the clinical link between reduced expression of
TET1, TIMP2, and TIMP3 and breast cancer progression.
Given that one of the pivotal functions of TIMP2 and TIMP3 is
to inhibit the activity of MMPs which in general promote cancer
invasion and metastasis (Clark et al., 2008;
Murphy, 2011), we
asked whether TET1 deficiency causes an increase in MMP
function. To this end, we performed reverse zymography
anal-ysis. As a control, only gelatin, the substrate of MMP2 and
MMP9, at the position of TIMP2 (
20 kD) and TIMP3 (24 kD)
remained intact and stained by Coomassie Blue (Figure 5D,
lane 1). Upon TET1 depletion, the level of gelatin at these two
specific positions was decreased (lane 2), presumably due to
the reduced level of TIMP2 and TIMP3 and the increased activity
of MMP2 and MMP9. This result strongly suggests that optimal
TIMP2 and TIMP3 activities require TET1. Consistently, TET1
knockdown stimulated the total MMP activity, as detected by
fluorescence generated by a peptide after cleavage by MMPs
(Figure 5E), indicating that TET1 expression is necessary for
the inhibition of MMP activity. Together, these experiments
uncover an essential role of TET1 in maintaining the expression
and function of TIMP2 and TIMP3.
TET1-Mediated Invasion Suppression Requires TIMP2
and TIMP3
To further study whether TIMP2 and TIMP3 directly contribute to
TET1-mediated suppression of cell invasion and metastasis, we
first ectopically expressed TIMP2 or TIMP3 in TET1-eliminated
cells, then performed a cell-invasion assay. As expected, the
exogenously expressed TIMP2 or TIMP3 significantly
down-regulated cell invasion induced by TET1 knockdown (Figure 6A),
indicating that expression of TIMP2 or TIMP3 is capable of
com-pensating for the lack of TET1 in invasion suppression. Next we
knocked down TIMP2 or TIMP3 to evaluate whether they
account for TET1 function in inhibiting invasion. Consistent
with previous results (Anania et al., 2011), depletion of TIMP2
or TIMP3 potentiated cell invasion (Figure 6B, bars 1–3).
Impor-tantly, TET1 elimination-induced cell invasion (bars 4 and 5) was
lost in cells without TIMP2 or TIMP3 (bars 6–9), indicating that
TIMP2 and TIMP3 are major factors responsible for TET1
func-tion in breast cancer cell-invasion suppression. Similar results
were observed in prostate cancer cells 22Rv1 (Figure S13A). In
another prostate cancer cell line, LNCaP, we observed that
50% and 70% of TET1-knockdown-induced cell invasion was
lost in cells when TIMP2 or TIMP3, respectively, was depleted
(Figure S13B; compare bars 3–6 with bars 1 and 2). Together,
these results suggest that TIMP2 and TIMP3 are likely to be
the major players responsible for TET1 activity in invasion
suppression. In LNCaP, TET1 may function through the
collabo-rated efforts of TIMP2 and TIMP3 as well as other invasion
medi-ators. Given the heterogeneity of cancers and the potential
redundancy in the pathways involved in cell invasion, it is not
surprising that subtle variation across different cell lines was
observed in this case.
TET1 Directly Binds to CpG-Rich Regions of TIMP2
and TIMP3 Genes and Inhibits Their DNA Methylation
After we established that TIMP2 and TIMP3 are important and
essential downstream targets of TET1 in invasion suppression,
the mechanism by which TET1 activates the expression of
TIMP2 and TIMP3 genes was further explored. Given that
TET1 is known to bind to CpG islands (Ito et al., 2010;
Williams
et al., 2011;
Wu and Zhang, 2011b;
Xu et al., 2011b;
Zhang
et al., 2010) and that both TIMP2 and TIMP3, but not TIMP1,
genes are enriched in CpG content, we examined the possibility
that TET1 may directly regulate TIMP2 and TIMP3 expression by
binding to their CpG islands. ChIP analysis demonstrated that
exogenously expressed Flag-tagged TET1 bound to the
CpG-rich regions of TIMP2 and TIMP3 genes, but not the upstream
distal site (Figures S14A and S14B). Consistently, the
endoge-nous TET1 bound to the TIMP3 gene through the CpG island,
but not other regions of the TIMP3 gene body or the regulatory
sequences of GADD45 (An et al., 2004) (Figure 7A). The
endog-enous TET1’s binding to the TIMP3 gene was specific, as the
ChIP signals within the CpG island were reduced to less than
A
B
C
D
E
Figure 5. TET1 Is Required for Expression and Function of TIMP2 and TIMP3
(A) Relative mRNA levels of TIMP1, TIMP2, and TIMP3 in indicated cells with TET1 shRNA (TET1-KD) compared to control.
(B) TIMP2 and TIMP3 mRNA levels positively correlate with TET1 expression in clinical breast cancer specimens. Of breast cancer patients, 41 representative patients from each group with the lowest and the highest expression levels of TET1 in the cancerous tissues (Figure S9) were analyzed for TIMP2 and TIMP3 mRNA levels with normalization to actin expression. The mean values are indicated.
(C) Low TET1 and TIMP2, or TIMP3 levels correlate with advanced node status in clinical breast cancer specimens. The same set of samples as in (B) was analyzed. 41 patients with high or low TET1 expression were split into two groups with high (n = 20) or low (n = 21) levels of TIMP2 or TIMP3 and were analyzed for node status as inFigure 4B.
50% when the total TET1 mRNA level was downregulated to
around 30% of the initial level by shRNA (Figure 7B). Our result
is consistent with previous reports in which TET1 was found to
be associated with the gene body, in addition to the gene
promoter region and the region around the transcription start
site (Williams et al., 2011;
Wu et al., 2011). Note that the relative
position of the transcription start site of the TIMP3 gene shown in
Figure 7A was defined according to NCBI prediction. Indeed, the
structure of TIMP3 promoter is poorly understood and the
posi-tion of its transcripposi-tion start site is controversial. Nevertheless,
previous reports have shown that the methylation status of the
TIMP3 gene region found in our study to be bound by TET1
correlates with TIMP3 gene expression (Bachman et al., 1999).
Thus, it is likely that TIMP3 has a noncanonical promoter that
is located downstream of the transcription start site. Together
with the data shown in
Figure 5, these results suggest that
TET1 potentiates the expression of TIMP2 and TIMP3 by
specif-ically binding to the CpG-rich sequences of TIMP2 and TIMP3
genes.
Given that TET1 is able to convert 5mC to 5hmC,
5-formylcy-tosine, and 5-carboxycytosine (5caC), which is then removed by
thymine DNA glycosylase (TDG)-coupled base excision repair
(He et al., 2011;
Ito et al., 2011), we analyzed whether TET1’s
binding to TIMP2 and TIMP3 genes alters the methylation status
of these two genes. As shown in
Figures 7C and
S14C, TET1
depletion increased and decreased TIMP2 and TIMP3
gene-associated 5mC and 5hmC, respectively. Note that in some
binding regions of these genes, the fold change of
TET1-loss-mediated 5mC increase was not exactly comparable to
the decrease of 5hmC. We reasoned that 5hmC could be
under-represented because of its conversion to 5caC, which is rapidly
removed by TDG. In the regions of amplicons 3 and 4 in M10 cells
(Figure 7C), TET1 depletion caused only significant decrease of
5hmC, but not increase of 5mC, suggesting that these DNA
regions were decorated with 5hmC instead of 5mC in the
pres-ence of TET1 and that these sites were not methylated when
TET1 was depleted.
Our results so far strongly suggest that cancer-cell-associated
hypermethylation of TIMP2 and TIMP3 genes reported
previ-ously by others (Bachman et al., 1999;
Smith et al., 2008) is
most likely due to the loss of TET1 during cancer development.
To further test this hypothesis, we treated MDA-MB-231 cells
with the DNA methylation inhibitor 5-aza-dC, then TIMP2 and
TIMP3 mRNA levels were evaluated. Consistent with earlier
studies (Anania et al., 2011;
Bachman et al., 1999), 5-aza-dC
significantly increased TIMP2 and TIMP3 expression (Figure 7D).
Notably, 5-aza-dC abolished TET1-depletion-mediated
sup-pression of TIMP2 and TIMP3 mRNA levels (Figure 7E). Given
that 5-aza-dC did not increase TET1 expression in these
exper-iments (data not shown) and that 5-aza-dC is known to reduce
the DNA methylation of TIMP2 and TIMP3 genes (Bachman
et al., 1999;
Cameron et al., 1999), these results support the
notion that TET1 regulates TIMP2 and TIMP3 expression through
its controlling of their DNA methylation level. Collectively, these
studies indicate that TET1 directly binds to CpG-rich regions of
TIMP2 and TIMP3 genes and downregulates their DNA
methyla-tion. Consequently, loss of TET1 in cancer cells most likely leads
to hypermethylation and shutdown of TIMP2 and TIMP3 genes.
Exciting evidence has indicated that TET1 plays an important
role in the suppression of global DNA methylation, maintenance
(D) TET1 depletion diminishes TIMP2 and TIMP3 activities. The medium collected from MDA-MB-231 cells with or without TET1 shRNA was analyzed by reverse zymography assays (seeExperimental Procedures). The relative intensity of gelatin in TIMP2 and TIMP3 positions was quantified with ImageJ (right). (E) TET1 knockdown increases MMP activity. The MMP activity of MDA-MB-231 cells with or without TET1 shRNA was analyzed by generic fluorogenic assay, quantified and shown as in (D).
Data in (A), (D), and (E) are shown with mean± SD from triplicate experiments. p values were measured by Student’s t test in (B–E). *p < 0.05; **p < 0.01; ***p < 0.001.
*
4
5
6
7
8
9
-
+ -
+ -
+
si-TIMP2
si-TIMP3
sh-TET1
Invasion-MDA-MB-231
B
0
1
2
3
0
0.5
1
1.5
0
0.5
1
1.5
1 2 3 4
A
sh-TET1
TIMPs
Rel
a
ti
v
e
cel
l index
Invasion-MDA-MB-231
-
+ + +
-0
1
2
3
*
*
**
Rel
ati
v
e cel
l index
Rel
ati
v
e cel
l index
0
1
2
3
4
1 2 3
**
**
Figure 6. TET1-mediated Invasion Suppression Requires TIMP2 and TIMP3
(A) Exogenous TIMP2 or TIMP3 expression suppresses cell invasion induced by TET1 depletion. MDA-MB-231 cells with TET1 shRNA were transfected with vector alone or expression plasmid for TIMP2 or TIMP3, followed by cell invasion assay.
(B) TET1 knockdown-induced cell invasion is lost by depletion of TIMP2 and TIMP3. MDA-MB-231 cells with or without TET1 shRNA were transfected with scramble RNA or siRNA against TIMP2 or TIMP3, followed by cell invasion assay. Data were collected at 42 hr post transfection of siRNA.
A
B
C
D
E
Figure 7. TET1 Binds toTIMP3 and Inhibits Its Methylation
(A) Endogenous TET1 binds to the CpG island of TIMP3. M10 cells were subjected to ChIP with PCR primers (distal, #1, #2, #3, #4, #5, and #6) against the indicated regions along TIMP3 shown in the diagram above the bar charts. TSS, transcription start site.
of mouse embryonic stem cell state (Ficz et al., 2011;
Ito et al.,
2010;
Tahiliani et al., 2009;
Wu et al., 2011), and mouse neuronal
activity (Guo et al., 2011). Here, we further provide the first
mechanistic evidence demonstrating that TET1 acts as a
cancer-invasion suppressor by inhibiting essential downstream
gene methylation. In summary, our data indicate that TET1
potentiates TIMP2 and TIMP3 expression, which in turn
down-regulates the MMP activity required for cell invasion. It is likely
that during or prior to cancer development, TET1 expression is
downregulated. Consequently, TIMP2 and TIMP3 levels are
reduced and MMPs are activated, followed by cell invasion
and metastasis. Recent findings point out that the TIMP family
proteins may have MMP-independent functions in inhibiting
cancer formation (Murphy, 2011). Therefore, TET1-mediated
TIMP2 and TIMP3 expression may have a broader impact on
cancer progression. Our work also highlights and provides
detailed data of the two described activities of TET1, its
enzy-matic activity and DNA-binding capability, in TET1-mediated
invasion suppression. In addition, we test our hypothesis that
TET1 is an invasion suppressor not only in vitro but also in vivo,
correlating TET1 expression levels with xenograft tumor
forma-tion and patient outcome. Interestingly, we found that TET1
depletion, in addition to stimulating cell invasion, promoted
prostate cancer cell proliferation and migration (Figures 2A
and
S3). In contrast, knocking down TET1 decreased normal
and cancerous breast cell growth and migration (Figures S5D
and S5E) but enhanced their invasion ability (Figure 3) in the
monolayer cell culture system. These results indicate that
TET1 most likely has a differential function in suppressing
cancer formation of different origins. The common step
regu-lated by TET1 in both prostate and breast cancers is ‘‘cell
invasion’’.
Consistent with our results, several recent reports show a
profound loss of TET gene expression and/or 5hmC in cancer
cell lines (Song et al., 2011) as well as in a variety of human
cancers such as breast, prostate, liver, lung, pancreatic,
colo-rectal, gastric, small intestine, brain, kidney, and skin cancers
(Haffner et al., 2011;
Jin et al., 2011;
Kudo et al., 2012;
Yang
et al., 2012). Indeed, loss of gene expression is not the only
way to compromise TET1 function in tumors. IDH1 and IDH2
mutations in glioma are found to generate oncometabolite
2-hydroxyglutarate, a known competitive inhibitor of the
2-ketoglutarate-dependent enzymes such as TET proteins (Xu
et al., 2011a). Together, these studies and ours strongly suggest
that loss of TET1-mediated invasion suppression is most likely
universal among human cancers. Additional studies using the
recently reported Tet1-deficient mice (Dawlaty et al., 2011) will
be useful in providing more decisive in vivo evidence in this
regard.
EXPERIMENTAL PROCEDURES
Prostate Cancer Samples and Immunohistochemistry
A total of 153 transurethral resection or prostatectomy specimens of prostate cancer, which received detailed pathological assessment and regular follow-up at the National Taiwan University Hospital, were selected for this study. The study was conducted according to the regulation of the ethics committee, and the specimens were anonymous and analyzed in a blinded manner. For immunohistochemistry, the formalin-fixed, paraffin-embedded tissue sections were deparaffinized and hydrated in a series of graded alcohol to water. Antigen retrieval was performed by incubation of the tissue sections with citrate buffer (DakoCytomation) at 125C for 5 min. Endogenous peroxidase activity was quenched with 3% H2O2and endogenous biotin was blocked with Background Buster (Innovex Biosciences). Tissue sections were then incubated with TET1 Ab (GeneTex 124207) diluted to a concentration of 1:1000 for 1 hr at room temperature and detected with the STAT-Q IHC staining system (Innovex Biosciences). After development with the substrate 3,3’ Diaminobenzidine, the slides were counterstained and mounted for light microscopy analysis.
Cell Migration and Invasion
Migration and invasion were measured with the Real-Time Cell Analyzer (RTCA) Dual Plate (DP) system (xCELLigence, Roche Diagnostics GmbH) and indi-cated by the cell index as defined inhttp://www.roche-applied-science.com/ sis/xcelligence/index.jsp?&id=xcect_010100. The system monitors cell status using proprietary microelectronic sensor technology. In brief, 23 104
to 53 104 of cells were seeded with serum-free medium onto the upper chambers of the cellular invasion/migration (CIM) plates. These chambers were then placed on the lower parts of the CIM device, which contained growth medium supplemented with 10% FBS as an attractant. As for invasion, upper chambers were coated with 20mg of matrigel (BD Biosciences, Cat No. 354234) for 4 hr before cells were added. Cell invasion and migration were monitored every 15 min for 48 hr.
Animal Experiments
Athymic (nu/nu) nude mice and BALB/c mice (6–7 weeks of age) were obtained from the National Laboratory Animal Center and housed as described previ-ously (Tsai et al., 2009). All animal work was performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee, Academia Sinica. For orthotopic implantation of human prostate cancer cells, a nude mouse prostate was exposed with a surgical incision and a suspension of prostate cancer cells (33 105in 20ml PBS) was injected into the left side of prostate. For breast xenograft tumor experiments, mouse mammary carcinoma 4T1 cells were orthotopically implanted into the fat pad of BALB/c mice. Biolu-minescence intensity of implanted tumors was monitored and body weight measured in living mice weekly. Mice were euthanized and necropsied at the end of experiments. A portion of each tumor was snap-frozen in liquid nitrogen and stored at 80C until needed for western blot analysis of relevant bio-markers. The remainder was fixed in 10% formalin overnight. For the acquisition of prostate cancer cells at different stages, tumors were harvested at 2 weeks or 15 weeks after implantation from prostate and lung tissues.
(B) Controls for TET1 Ab used in the ChIP assays shown in (A). ChIP assays similar to (A) were performed in M10 cells with scramble shRNA (black bars) or TET1 shRNA (gray bars).
(C) TET1 knockdown increases and decreases TIMP3 promoter-specific 5mC and 5hmC levels, respectively. M10 or MDA-MB-231 cells with or without TET1 shRNA were subjected to MeDIP analysis with 5mC or 5hmC Ab. PCR was carried out with four independent pairs of primers with the relative positions indicated in (A). Antibodies used are Flag Ab (a kind gift from Dr. S.C. Lee), TET1 Ab (GeneTex, #124207), IgG (Abcam), 5mC Ab (Eurogentec, BI-MECY-0100), and 5hmC Ab (Active Motif, #39769).
(D) 5-aza-dC derepresses the expression of TIMP2 and TIMP3.
(E) TET1-depletion-mediated repression of TIMP2 and TIMP3 is lost in the presence of 5-aza-dC.
(D and E) MDA-MB-231 cells without (D) or with (E) TET1 shRNA or scramble shRNA were mock treated or treated with 5-aza-dC, followed by real-time RT-PCR analysis of TIMP2 and TIMP3 gene expression. In (C–E), p values were measured by Student’s t test. **p < 0.01; ***p < 0.001. In (A–E), data are shown as mean± SD from triplicate experiments.
Human Breast Cancer Specimens
All breast tissue specimens are from the BioBank of the Tri-Service General Hospital. All patients gave informed consent for participation, and the study was approved by the institutional review board of the Tri-Service General Hospital. These patients include individuals with carcinoma in situ and invasive ductal carcinoma of the breast who are undergoing mastectomy and axillary lymph node dissection. Stage was determined according to the AJCC system (American Joint Committee on Cancer,http://www.cancerstaging.org/). All specimens, including cancerous parts and adjacent normal parts, were collected during operations and subsequently stored in liquid nitrogen.
Reverse Zymography and MMP Assays
A total of 53 105
of MDA-MB-231 cells were seeded onto 6-well plate. After 24 hr, the cells were washed and incubated with serum-free medium for 48 hr. The medium (defined as conditioned medium, CM), was then collected and centrifuged at 10003 g for 10 min, followed by zymography assays (Clark et al., 2010). In brief, CM was mixed with nonreducing 53 sample buffer without boiling and was loaded onto 15% SDS-PAGE containing 0.1% gelatin and serum-free medium after being cultured with M10 cells. Electrophoresis was performed at 125 V for 120 min. The gel was then removed and rinsed twice in enzyme-renaturing buffer (2.5% of Triton X-100, 50 mM Tris-base, 200 mM NaCl, 5mM ZnCl, 25 mM CaCl2[pH 7.5]) for 30 min with gentle agita-tion at room temperature (RT), followed by incubaagita-tion in the developing buffer (50 mM Tris-base, 200 mM NaCl, 5mM ZnCl, 25 mM CaCl2[pH 7.5]) for 30 min with gentle agitation at RT and again in the same buffer for 16–18 hr at 37C. After being washed three times with deionized water, the gel was stained by coomassie blue staining. Quantification was performed with ImageJ as described previously (Hu and Beeton, 2010). For MMP assays, a SenoLyte 520 Generic MMP Assay Kit (AnaSpec) was used according to the instructions provided by the manufacturer. In brief, CM was mixed with an equal volume of MMP substrate solution in the 96-well black microplate and incubated for 1 hr at RT. The fluorescence intensity was measured by a Victor 3 1420 multilabel counter (Perkin Elmer) at Ex/Em = 540 nm/575 nm.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation assays were performed as described previ-ously (Hsu et al., 2004) except that the eluted DNA was extracted via a PCR purification kit (QIAGEN). DNA was then analyzed by real-time qPCR (LightCycler 480, Roche). The amplifications were performed in a reaction volume of 20ml containing 2 ml of immunoprecipitated material. The sequences of DNA primers for ChIP are listed in the Extended Experimental Procedures.
Methylated DNA Immunoprecipitation
Genomic DNA was prepared with a genomic DNA extraction kit (QIAGEN, #51304) and sonicated with Bioruptor (Diagenode) to produce random frag-ments ranging in mean size from 300 to 1,000 bp. 5mg of fragmented DNA was denatured for 10 min at 95C and immunoprecipitated overnight at 4C with 5ml of 5-methylcytidine antibody (Eurogentec) in a final volume of 500ml IP buffer (10 mM sodium phosphate [pH 7.0], 140 mM NaCl, 0.05% Triton X-100). The mixture was incubated with 30ml magnetic beads (Milipore) for another 4 hr at 4C and washed three times with 1 ml of IP buffer. Beads were resuspended with 250ml digestion buffer (50 mM Tris [pH 8.0], 10 mM EDTA, 0.5% SDS) containing 5ml proteinase K (20 mg/ml stock) and shaken overnight at 56◦C. DNA was extracted with a QIAGEN Kit (QIAGEN, #28106) and analyzed by real-time PCR.
5-aza-dC Treatment
The demethylation agent 5-aza-dC was added to the culture medium at the concentration of 10mM. Cells were harvested and RNAs analyzed by quan-titative real-time RT-PCR 5 days after 5-aza-dC treatment.
For other methods, please see theExtended Experimental Procedures.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures and 14 figures and can be found with this article online athttp://dx.doi.org/10. 1016/j.celrep.2012.08.030.
LICENSING INFORMATION
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 Unported License (CC-BY-NC-ND;http://creativecommons.org/licenses/by-nc-nd/3.0/ legalcode).
ACKNOWLEDGMENTS
We thank Drs. W.H. Lee at UCI and Y. Zhang at UNC for critical suggestions, S.C. Lee at NTU for providing the Flag antibody, and Y.C. Chang at NTUH for helping with statistics and analyzing patient survival. We also thank the National RNAi Core Facility at Academia Sinica for providing shRNA constructs and Affymetrix Gene Expression Service Laboratory at Academia Sinica for performing the microarray experiments. This research was primarily supported by a career development grant from Academia Sinica to L.-J.J. and partially supported by National Science Council grants to P.-W.H. (NSC 98-2320-B-001-018-MY) and H.-D.H. (NSC-100-2911-I-009-101).
Received: May 12, 2012 Revised: July 12, 2012 Accepted: August 31, 2012
Published online: September 20, 2012
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