Silencing of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs Stimulates Hyperplastic Phenotypes through Activation of Epidermal Growth Factor Receptor and Hypoxia-Inducible Factor-2α
et al. (2013) Silencing of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs
Stimulates Hyperplastic Phenotypes through Activation of Epidermal Growth Factor Receptor and Hypoxia-Inducible Factor-2. PLoS ONE 8(12): e84520.
Silencing of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs Stimulates Hyperplastic Phenotypes through Activation of Epidermal Growth Factor Receptor and Hypoxia-Inducible Factor-2
You Mie Lee 0
Sun-Hee Lee 0
Kheun Byeol Lee 0
Minh Phuong Nguyen 0
Min-Young Lee 0
Gyu Hwan 0
Mi Jeong Kwon 0
Peiwen Fei, University of Hawaii Cancer Center, United States of America
0 1 Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University , Daegu , Republic of Korea, 2 School of Life Sciences and Biotechnology, Kyungpook National University , Daegu , Republic of Korea
Reversion-inducing cysteine-rich protein with Kazal motifs (RECK, a tumor suppressor) is down-regulated by the oncogenic signals and hypoxia, but the biological function of RECK in early tumorigenic hyperplastic phenotypes is largely unknown. Knockdown of RECK by small interfering RNA (siRECK) or hypoxia significantly promoted cell proliferation in various normal epithelial cells. Hypoxia as well as knockdown of RECK by siRNA increased the cell cycle progression, the levels of cyclin D1 and c-Myc, and the phosphorylation of Rb protein (p-pRb), but decreased the expression of p21cip1, p27kip1, and p16ink4A. HIF-2 was upregulated by knockdown of RECK, indicating HIF-2 is a downstream target of RECK. As knockdown of RECK induced the activation of epidermal growth factor receptor (EGFR) and treatment of an EGFR kinase inhibitor, gefitinib, suppressed HIF-2 expression induced by the silencing of RECK, we can suggest that the RECK silenicng-EGFR-HIF-2 axis might be a key molecular mechanism to induce hyperplastic phenotype of epithelial cells. It was also found that shRNA of RECK induced larger and more numerous colonies than control cells in an anchorage-independent colony formation assay. Using a xenograft assay, epithelial cells with stably transfected with shRNA of RECK formed a solid mass earlier and larger than those with control cells in nude mice. In conclusion, the suppression of RECK may promote the development of early tumorigenic hyperplastic characteristics in hypoxic stress.
Funding: This work was supported by the National Research Foundation (NRF) grant funded by the Korean government (MSIP)
(NRF-2012R1A4A1028835). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Reversion-inducing cysteine-rich protein with Kazal motifs
glycoprotein that contains multiple epidermal growth
factor(EGF)-like repeats and multiple serine protease inhibitor-like
domains . RECK inhibits proMMP-2 activation and the
enzymatic activities of MMP-2, MMP-9, and membrane type 1
(MT1)-MMP [2,3]. Although RECK functions as an inhibitor of
matrix metalloproteinases (MMPs), it does not share structural
homology with tissue inhibitors of metalloproteinases (TIMPs)
. RECK is expressed in various normal human tissues, but
breast, lung, colorectal, prostate, and gastric cancer, or in
osteosarcomas . RECK downregulation in cancer tissues is
associated with a low survival rate and a poor prognosis
because RECK inhibits angiogenesis, invasion, and metastasis
in cancer via MMP inhibition . Germ line knockout of RECK
induced the proliferation of mouse embryonic fibroblasts
(MEFs) and enabled early escape from the cellular senescence
induced by oncogenic insults, and RECK interferes with
epidermal growth factor receptor (EGFR) signaling .
Epigenetics, such as, histone and DNA modifications, are
involved in the silencing of RECK [6,7]. We previously reported
that RECK is downregulated under hypoxic conditions through
has not been detected in oncogenically transformed cells and
in various type of cancers, such as, hepatoma, pancreatic,
microRNAs are also involved in targeting RECK in hypoxia and
RAS-mediating signaling pathways . Thus, it has been
suggested that HIF-1 is a key regulator to inhibit RECK
expression in tumorigenesis [8,9].
Hypoxia (a reduction in tissue oxygen tension) is frequently
detected in growing tumors larger than 1 mm3 .
Interestingly, in inflamed tissues, oxygen concentrations are
often far below physiological levels . Hypoxia results in
adaptive changes to the transcriptions of a wide range of genes
involved in increasing oxygen availability to tissues and
decreases the cellular consumption of oxygen such as
angiogenesis or glycolysis or apoptosis via HIFs- .
Differential activity and expression pattern of HIF-1 and
HIF-2 in the tissues have been well identified . In cell cycle
progression, stabilization of HIF-1 results in cell cycle arrest
due to the inhibition of the transcriptional activity of c-Myc. On
the other hand, HIF-2 exhibits opposing effects that is, it
increases cell proliferation by activating c-Myc .
Many reports have investigated that hypoxia attenuates the
expressions of tumor suppressor genes, such as, p53, von
Hippel Lindau (VHL), MLH1, BRCA1, 2, and RUNX3 including
RECK in normal and cancer cells [8,9,14-17]. The products of
these tumor suppressor genes primarily function as
gatekeepers of cell proliferation, and thus, loss of function or
silencing of the tumor suppressors play critical roles in the
processes that permit the unregulated proliferation and
transformation of normal cells under precancerous hypoxic
conditions [18-20]. However, biological functions and
significances of RECK silencing under hypoxic conditions in
hyperplastic phenotypes of early tumorigenesis have largely
Here we demonstrate that RECK silencing under hypoxic
conditions induced cell proliferation in normal epithelial cells
and their tumorigenic potential such as anchorage-independent
colony forming ability. Knock-down of RECK expression by
siRNAs increased c-Myc-mediated cell cycle progression. Our
results also suggest that RECK might be an upstream regulator
that suppresses HIF-2 through EGFR in obtaining an early
tumorigenic hyperplastic phenotype. Therefore, our data reveal
a novel mechanism of RECK and hypoxic conditions for the
induction of hyperplastic cells in an early step of tumorigenesis
through HIF-2 and EGFR.
Materials and Methods
Ethnics Statement and Chemicals
Animal care and experimentation were performed in
accordance with procedures approved by the KNU (Kyngpook
National Unversity) Animal Care and Use Committee. An
inhibitor for MMP-2 and -9 (#444241), an ERK MAPK inhibitor,
PD98059 were purchased from Merck Millipore, and gefitinib,
an EGFR specific inhibitor was purchased from Cayman
Chemical (Ann Arbor, MD).
Cell culture and hypoxic incubation
HEK293 human embryonic kidney and TCMK mouse kidney
(American Type Culture Collection, Manassas, VA) cells were
maintained in DMEM supplemented with 10% fetal bovine
serum (FBS, Hyclone, Logan, UT) and 1X antibiotics (100
units/ml penicillin, 100 g/ml streptomycin, both from
Invitrogen, Carlsbad, CA). MCF10A normal human breast
epithelial cells were maintained in DMEM/F12 supplemented
with insulin, cholera toxin (Sigma, St Louis, MO), rEGF (R&D
systems, Wiesbaden, Germany), hydrocortisone, and
Lglutamine (Invitrogen, Gaithersburg, MD). PrEC prostate
epithelial cells (Clonetics Cambrex Corp, Walkersville, MD)
were cultured in keratinocyte growth media (Lonza, Basel,
Switzerland) containing 10% FBS and 1X antibiotics. Cultures
were maintained under either hypoxic (1% O2, 5% CO2 and
balanced with N2) or normoxic (21% O2, 5% CO2 and balanced
with N2) conditions at 37C (Thermo Scientific, MA, USA).
siRNA experiment and transfection
Two double strand siRNAs designed to target RECK
(Qiagen, Valencia, CA), HIF-1, and HIF-2 and a scrambled
siRNA were synthesized (Bioneer, Daejeon, Korea). HEK293
cells were transfected with double strand RNA using HiPerFect
Transfection reagent (Qiagen) according to the manufacturer's
protocol. RECK mRNA expression was evaluated by RT-PCR
and RECK protein expression was monitored by western
blotting at the indicated times post-transfection. Proliferation
assays were performed on 96 well after splitting cells at 24 h
post-transfection. SiRNA1 was designed to target
5CAGATTGAAGCCTGCAATAAA-3, and siRNA2 was designed
to target 5-ATACCTGTTCTTGATATTAAA-3. pBluescript
RECK full-length plasmid (Invitrogen) was cloned into pCMV5
vector after digestion with KpnI and SacI. Plasmids were
transfected into HEK293 cells that had been plated at 1x106
cells per 60 mm dish one day previously. Transfections were
performed using Lipofectamine 2000 reagent (Invitrogen)
according to the manufacturers instructions. At 24 h
posttransfection, cells were plated onto 96-well plates for
proliferation assays or onto 60 mm dishes for protein collection.
Western blot analysis
Equal amounts of protein extracts in SDS-lysis buffer were
subjected to SDS-PAGE analysis and then transferred to
nitrocellulose membranes. We used the following human
antibodies: anti-RECK (BD Bioscience, San Diego, CA), cyclin
D1, cyclin A, p27, NFkB (Santa Cruz Biotechnology, Santa
Cruz, CA), p21 (Cell signaling), phosphorylated EGFR, ERK1/2
(Cell Signaling), EGFR (Santa Cruz Biotechnology, Santa
Cruz, CA), -actin, and -tubulin (Sigma, St Louis, MO). An
enhanced chemiluminescence system (Pierce, Woburn, MA)
was used for detection. Antibody for pEGFR may cross-reacts
slightly with other EGFR family member (eg. ErbB2) or
activated PDGF receptor.
Cell proliferation assay and cell cycle analysis
Cell proliferation assays were performed by using Cell
Counting Kit-8 (CCK-8) solution (Dojindo, Gaithersburg, MA).
Cells (5 x 103) were seeded onto a 96-well plate. After 24 hours
(time 0), we started to measure cell proliferation at the
indicated time points. When we seeded 5 x 103 cells in wells,
one day later densities became around 20%. Because the cell
proliferation rate in hypoxia was higher than in normoxia, only
hypoxic cells were almost confluent (- 95%) after 72 h of
incubation. The graph represents fold changes in numbers of
cells at each time versus control cell number at time 0. Cells
were plated at 1x105 per 60 mm dish and exposed to normoxic
or hypoxic condition for the indicated times. The DNA contents
and cell cycle statuses of permeabilized cells were determined
by propidium iodide (PI, Sigma-Aldrich) staining. Stained cells
were processed using a fluorescence-activated cell sorter
(FACS, Coulter Elite ESP Cell Sorter, Beckman) and cell cycle
profiles were analyzed.
Soft agar colony formation assay
Soft agar colony formation assays were performed using the
CytoSelect 96-well Cell Transformation Assay kit (Cell Biolabs,
San Diego, CA). Ten days after seeding, the numbers and
morphologies of colonies were determined using an inverted
phase-contrast microscope (Olympus, Japan). To quantify
anchorage-independent growth, colonies were lysed with lysis
buffer and detected with CyQuant GR dye. Fluorescence was
measured using a fluorometer and a 485/520 filter set (Wallac
Victor3 1420 mutilabel counter, PE). Data are presented as the
means SDs of three independent wells.
RECK shRNA stable transfection
To establish RECK shRNA stable transfectants, we
synthesized shRECK oligonucleotides harboring the RECK
siRNA1 sequence and cloned them into a blasticidine-resistant
pBLOCK-iT Gateway vector (Invitrogen), according to the
manufacturer's instructions. For a control vector, we used a
lamin shRNA entry clone and used the same procedure used
for the cloning of RECK shRNA into the destination vector.
shRNA transfectants were selected by treating cells with
blasticidine (5 g/ml) for 21 days. Cells stably transfected with
shRECK or shlamin were established and treated weekly with
blasticidine (5 g/ml) in DMEM containing 10% FBS.
Animal experiments and immunohistochemistry
Balb/c-nu mice were purchased from the Institute of Medical
Science, University of Tokyo (Tokyo) and maintained in a
specific pathogen free facility in accordance with the guidelines
issued by the Animal Care and Use Committee of Kyngpook
National University. Animals were provided with autoclaved tap
water and lab chow ad libitum and were housed in a 23
0.5C, 55 10 % relative humidity environment under a 12
hour light-dark cycle. For tumorigenesis experiments, 1x107
cells stably transfected with shRECK or shlamin were
suspended in 0.2 ml of Matrigel and injected into both flanks of
nude mice. Tumor growths were determined by measuring the
dimensions of tumors with a caliper every two days and
multiplying tumor height x length x depth (mm3). Tumor tissues
were fixed in 4% paraformaldehyde (pH 7.4) and embedded in
paraffin or OCT. Serial sections (5 m) were mounted on
polyL-lysine coated slides, and processed for either histology or
immunohistochemistry. Sections were immunostained with
antibodies against mouse CD31 (BD, NJ), p-pRb, PCNA, p16,
CA9, RECK (Cell Signaling), and a hypoxic probe (Chemicon
International), and visualized using appropriate
biotinconjugated secondary antibodies followed by
immunoperoxidase detection (Vectastain ABC Elite kit; Linaris,
Germany) using diamino-benzidine (DAB) as substrate (Vector,
U.K). Counterstaining was performed with hematoxylin.
Hypoxic probe-1 (pimonidazol HCl, Chemicon International)
was injected 4 h before tumor excision.
Hypoxia stimulated proliferations of human epithelial
To identify whether the downregulation of RECK is involved
in regulation of cellular growth or not, we blocked RECK
expression using siRNA and performed cell proliferation assays
in HEK293 kidney epithelial cells under normoxic conditions.
RECK mRNA and protein levels were clearly diminished at 48
h after transfection with two different siRNAs (Figure 1A).
Downregulation of RECK by siRNAs clearly increased cell
growth with time, and cell growth rate was inversely correlated
with the degree of RECK suppression by siRNA 1 (by 3.5 fold)
or 2 (by 2.9 fold) (Figure 1B).
Previously, we found that RECK was silenced at the
transcriptional level by HDAC and HIF-1 in HEK 293 cells
under hypoxic conditions . When we examined RECK
protein levels in four types of normal epithelial cells isolated
from specific tissues, HEK293 (human kidney), PrEC (human
prostate), TCMK (mouse kidney), and MCF10A (human breast)
after exposed to hypoxia, we observed RECK expression was
decreased in a time-dependent manner under hypoxic
conditions (Figure 1C). Exposure to hypoxia for 6 h significantly
stimulated the proliferations of HEK293, PrEC, TCMK, and
MCF10A cells. As compared with cells maintained under
normoxic conditions, proliferations increased by 2.0-3.2 fold
after 12-16 h of hypoxia and by 2.3-4.0 fold after 24 h (Figure
1D), when proliferations maintained a steady state due to
confluent cell densities. Because the cell proliferation rate in
hypoxia was higher than in normoxia, only hypoxic cells were
almost confluent (-95%) after 72 h of incubation. We restored
RECK expression with a full-length RECK-pCMV5 plasmid, and
then found that cell proliferation was significantly inhibited
under hypoxia (Figure 1E). These results strongly suggest that
hypoxia-induced RECK downregulation is responsible for
obtaining hyperplastic properties of normal epithelial cells.
Figure 1. RECK silencing induced by hypoxia stimulated epithelial cell proliferation. (A) Site and silencing efficiency of two
RECK siRNAs. The upper panel shows RECK siRNA sites (red bar). The lower panel shows that transfection of RECK siRNAs (5
nM) into HEK293 cells effectively blocked RECK expression. The experiment was performed in triplicate. (B) Proliferation assays
were performed at 24 h post-transfection (0 time) at the indicated times after seeding siRNA-transfected cells at 5x103 per well in
96-well plates. Fold change is the ratio of the cell population at each time point versus the start time (point 0). Data are presented as
means SDs (n=4). *, p<0.01 versus scrambled-transfected controls. (C) Total protein lysates were extracted from HEK293, PrEC,
TCMK, and MCF10A cells after exposure to normoxia or hypoxia for the indicated times. RECK expressions were determined by
western blotting. -actin was used as an internal control. (D) HEK293, PrEC, TCMK, and MCF10A cells were plated at a density of
5x103 in 96-well plates. After 24 h, cells were maintained under normoxic (N) or hypoxic (H) conditions for the indicated times. Cell
proliferation assays were performed using a CCK-8 cell proliferation assay kit. Data are presented as means SDs (n=4). *, p<0.01,
**, p<0.001 versus normoxic controls. (E) Full-length pCMV5-RECK plasmids were transfected into HEK293 cells, and RECK
protein levels and cell proliferation rate were determined by Western blot analysis and CCK-8 cell proliferation assay kit,
respectively, after incubation under normoxic (N) or hypoxic (H) conditions for 24 h. Data are presented as means SDs (n=3). *,
p<0.01, versus normoxic controls, **, p<0.01 versus hypoxic controls. All experiments were performed at least three times.
RECK downregulation induced by hypoxia was
responsible for cell proliferation
We next measured the levels of various cell cycle proteins
under hypoxic conditions and in parallel with the silencing of
RECK in HEK293 cells. Levels of cyclin D1, cyclin A, and
phosphorylated-Rb (p-pRb), as well as c-Myc (a critical factor
for G1-S transition) were increased both by hypoxic conditions
and by the silencing of RECK (Figure 2A, B). Consistently,
levels of p21cip1, p27kip1 and p16ink4A that inhibit cyclin/CDK
activity at the G1-S transition point, were depressed under
hypoxic conditions and by the silencing of RECK (Figure 2A, B)
and these results are in-line with our observations of increased
cell proliferation shown in Figure 1. These results can suggest
that hypoxia-induced RECK downregulation is responsible for
obtaining hyperplastic properties of normal epithelial cells. To
determine if hypoxia-induced cell proliferation was due to a
decrease in cell death and/or an increase in cell cycle
Figure 2. Hypoxia and silencing of RECK increased cell cycle progression. (A) Western blot analysis with anti-c-Myc, cyclin
D1, cyclin A, phosphorylated Rb (p-pRb), p21, p27, and p16 were performed under normoxic (N) and hypoxic (H) conditions after 24
h of exposure. (B) After transfecting scrambled siRNA (Scr) and two kinds of siRECK (si1 and si2), levels of cell cycle proteins in
cell lysates were determined by Western blotting. (C) Cell cycle analysis by FACS after PI staining. The diagram shows the diploid S
and sub-G1 phase HEK293 cells under hypoxic conditions up to 72 h after staining and those of normoxic controls (cont). Right
cytograms show the proportion of cell populations in each cell cycle stage in 12 h normoxia (12N) and hypoxia (12H). All
experiments were performed at least in duplicate.
progression, we subjected HEK293 cells to flow cytometry.
Cells cultured under hypoxic conditions for 8 h to 72 h showed
1.6 fold increases in the proportions of cells in S phase as
compared with cells cultured under normoxic conditions. This
increase in the proportion of cells in the S phase continued
(with some fluctuation) over 72 h of hypoxic exposure (Figure
2C, see the dashed area for diploid S phase in 12 h hypoxia).
Cell death was monitored by counting subG1 phase
populations, and the measurements obtained suggested that
hypoxia did not induce cell death (Figure 2C). These results
suggest that an increase the S-phase transition and a lack of
change in cell death collaborate to stimulate cell viability and
proliferation under hypoxic conditions.
HIF-2 was involved in proliferation of epithelial cells
under hypoxic conditions induced by RECK silencing
Because HIF-1 and HIF-2 differentially regulate cell cycle
progression under hypoxia, we checked the expressions of
HIF-1 and HIF-2 under hypoxic and RECK- knockdown
conditions, hypoxia increased HIF-1 and HIF-2 protein
levels. However, although RECK knockdown induced the
upregulation of HIF-2 expression, it did not upregulate HIF-1
(Figure 3A), which suggested RECK is an upstream regulator
of HIF-2 but not of HIF-1. Surprisingly, NFB and
phosphorylated STAT1 was also upregulated by RECK
knockdown and hypoxia (Figure 3A). HIF-2 mRNA was
upregulated by RECK knockdown and hypoxia (Figure 3B),
suggesting RECK regulates HIF-2 at the transcriptional level.
Hypoxia-induced cell proliferation was blocked in HIF-1
and/or HIF-2 silenced cells from 6 h to 24 h of culture under
hypoxic conditions (Figure 3C). We found HIF-1 and HIF-2
siRNAs were effective and confirmed that silencing of HIF-1
inhibited RECK mRNA expression but silencing of HIF-2 did
not. Furthermore, the mRNA expressions of HIF-1 and/or
HIF-2 under hypoxic conditions were unchanged (Figure 3C,
After 24 h of culture under hypoxic conditions,
hypoxiainduced cell proliferation was blocked in HIF-1 and/or HIF-2
silencing cells (Figure 3C). The results may explain that HIF-2
is involved in the cell proliferation mediated by RECK-silencing
whereas HIF-1 is directly involved in RECK gene silencing at
the transcriptional level .
Figure 3. The involvement of HIF-2 in hypoxia-induced RECK silencing-mediated cell proliferation. (A) HEK293 cells were
transfected with scrambled or RECK siRNA and exposed to normoxic or hypoxic conditions for 24 h. The expressions of HIF-1
HIF-2, NFB, and phophorylated-STAT1 were determined by western blotting. -actin was used as an internal control. (B) Using
the same samples as in A, the mRNA expressions of RECK and HIF-2 were determined by semi-quantitative RT-PCR. -actin was
used as an internal control. (C) HEK293 cells were plated onto 96-well plates and transfected with siHIF-1 and/or siHIF-2 or
scrambled siRNA using HiPerFect Transfection reagent (Qiagen). After 24 h, transfected cells were incubated under normoxic or
hypoxic conditions for an additional 24 h. At the indicated time points, cell proliferation assays were performed using CCK-8
solution. Data are presented as means SDs (n>3). Expression of HIF-1 and HIF-2 was confirmed in each siRNA transfectant by
western blot analysis (right panel). RECK, HIF-1, HIF-2 mRNA expression pattern was confirmed by semiquantitative RT-PCR at
indicated time point. -actin was used as an internal control (lower panel). *, p<0.01 significantly different from the normoxic control
group at each time point. , p<0.01 significantly different from the hypoxic control group at each time point.
Involvement of EGFR signaling in the HIF-2
expression induced by RECK silencing
The fact that RECK inhibits the EGFR signaling pathway 
made us to examine whether the inhibition of EGFR influences
HIF-2 expression induced by RECK silencing. After we
confirmed that HIF-2 was upregulated under hypoxia in a
time-dependent manner (Figure 4A, upper panel), we treated
siRECK-transfected or control cells with gefitinib (Ge, a
selective EGFR inhibitor) and PD98059 (PD) under normoxia
or hypoxia. Gefitinib suppressed RECK silencing- or
hypoxiainduced HIF-2 expression, and PD98059 (a MEK inhibitor)
had the same effect as gefitinib on HIF-2 expression (Figure
4A, lower panel). To confirm this result, we checked the
activation of EGFR and of its downstream molecule, p42/44
ERK MAPK, which is activated and involved in RECK silencing
in hypoxia , and found that hypoxia and RECK silencing
both activated EGFR and p42/44 ERK MAPK (Figure 4B). In
addition, we further investigated whether EGFR activation is
regulated by MMP or by the silencing of HIF-1 or HIF-2.
Levels of phosphorylated EGFR and downstream
phosphorylated ERK were unchanged by MMP inhibitor (Mib)
or siHIF-2 under hypoxia, but changed by siHIF-1 (Figure
4C), indicating the pathway responsible for MMP inhibition
differs from required for EGFR activation, and that EGFR
activation by RECK silencing is a downstream of HIF-1 and
upstream of HIF-2. These results indicate that the expression
of HIF-2 is induced by silencing of RECK via activation of the
EGFR signaling pathway.
Figure 4. Involvement of the EGFR signaling pathway in HIF-2 expression in RECK-silenced cells. (A) HEK293 cells were
exposed hypoxia for 6, 12, and 24 h and determined time-dependent HIF-2 expression by western blot analysis (upper panel).
HEK293 cells transfected with scrambled si RNA (-) or siRECK were treated with gefitinib (Ge, 1 M, an EGFR inhibitor) or
PD98059 (PD, 50 M; an ERK MAPK inhibitor) under normoxia (N) or hypoxia (H) for 24 h, and the protein expressions of HIF-2
were determined by western blot analysis. -actin was used for internal control. (B) The activations of EGFR and ERK-MAPK in
scrambled (scr) or siRECK (siR) transfected cells exposed to normoxia (N) or hypoxia (H) were determined by western blotting
using antibodies against total form or phosphorylated-form of EGFR or ERK. (C) The activations of EGFR and ERK and RECK
expression in siHIF-1 (siH1) -, siHIF-2 (siH2) -, and MMP inhibitor (Mib)-treated cells under hypoxia (H) were determined by
western blot analysis using antibodies against phosphorylated-EGFR and RECK antibody. Scr. Scrambled siRNA.
MMP activation was involved in hypoxia-stimulated
RECK functions as an inhibitor of MMP activation [22,23].
Therefore, we examined MMPs and MT1-MMP by zymography
and western blotting under hypoxic conditions. Hypoxia and
siRECK transfection significantly induced pro- and active
MMP-9 and MMP-2 by zymography, and increased MT1-MMP
protein expression levels (Figure 5A).
Cell proliferation was increased by hypoxia and by RECK
siRNA transfection but treatment with the MMP inhibitor
decreased cell proliferation induced by hypoxia or siRECK
transfection (Figure 5B). These findings suggest that MMP-2
and MMP-9 are involved in the activation of cell proliferation
induced by the silencing of RECK under hypoxic conditions, but
that the pathway involves differs from that of
EGFR-HIF-2induced cell proliferation.
Silencing of RECK gene promoted in vitro and in vivo
To confirm that the downregulation of RECK promotes the
hyperplastic activity of epithelial cells in vitro, we used soft agar
colony formation assays . Cells transfected with RECK
siRNA formed more colonies than those transfected with
scrambled siRNA (Figure 6A, a & c vs. b & d), and the
fluoroactivities of colony lysates from RECK siRNA transfected
cells were significantly higher than those of control siRNA
transfectants (Figure 6A, graph).
To explore the role of RECK downregulation in hyperplastic
development during early tumorigenesis, we generated stable
cells with RECK-knockdown. These cells were then injected
s.c. into nude mice flank, and tumor masses were measured
from 7 days post-injection for 22 days. As early as one week
post-injection, nude mice injected with shRECK transfectants
bore tumors with greater mass volumes and growth compared
to those injected with control cells (Figure 6B). Tumor
microvessel density assayed by CD31 immunostaining showed
that RECK knockdown was associated with highly vascularized
tumors (Figure 6C, a-b). Furthermore, levels of p-pRb and of
proliferating cell nuclear antigen (PCNA) were markedly higher
in shRECK tumors, but p16INK4A expression was completely
abolished (Figure 6C, c-h). To confirm the inverse relation
between hypoxia and RECK expression in tumors, we
examined hypoxic regions in RECK silenced tumors by
injecting pimonidazole (a hypoxic probe).
Immunohistochemistry showed that hypoxic regions (Figure
5D) and carbonic anhydrase IX (CA9) (a hypoxic marker
protein) expressions were greater in RECK silenced tumors
(Figure 6D). Taken together, these results suggest that RECK
suppression by hypoxia promotes the acquisition of a
premalignant hyperplastic phenotype and enhances in vitro and
in vivo tumorigenesis via G1/S phase cell cycle progression
through the activation of EGFR and HIF-2.
In this study, RECK expression was found to be
downregulated by hypoxia in normal epithelial cells and to be
involved in the development of the hypoxia-induced
hyperplastic phenotype. RECK silencing under hypoxic
conditions enhanced cell cycle progression through c-Myc, and
silencing of RECK induced EGFR activation and subsequently
HIF-2 expression. Colony formation assays and in vivo tumor
xenograft experiments suggested that the suppression of
RECK by hypoxic stress is required to transform normal cells to
Figure 5. MMP-2/-9 and MT1-MMP were also involved in hypoxia-induced epithelial cell proliferation. (A) Cells were
transfected with scrambled siRNA or the two types of siRECKs. After 24 h, transfected cells were incubated under normoxic or
hypoxic conditions for an additional 24 h. Conditioned media were collected from siRECK transfected (si1 and si2) or
hypoxiaexposed HEK 293 cells, and gelatin zymography was performed. Western blotting for MT1-MMP was also performed using protein
lysates obtained under each condition. (B) HEK293 cells were pretreated with a MMP inhibitor (5 M) 2 h before normoxic or
hypoxic incubation and cell proliferation assays were performed at the indicated times under normoxic (N) or hypoxic (H) conditions.
Data are presented as means SDs (n=4). *, p<0.01 versus the t = 0 control, **, p<0.01 versus the vehicle control.
tumor-like cells by enhancing their proliferative abilities.
Therefore, loss of RECK expression by microenvironmental
hypoxic stress may stimulate to get a more hyperplastic
phenotype in normal epithelial cells and to favor early
tumorigenesis events. Our results support previous
suggestions that RECK functions as a transformation
suppressor gene in premalignant and normal tissues [3,23].
We found although hypoxia stabilized HIF-1 and HIF-2,
only HIF-2 was upregulated by silencing of RECK, and that
knockdown of either HIF-1 or HIF-2 recovered
hypoxiainduced cell proliferation (Figure 3). Because HIF-1
participates in RECK downregulation at the transcription level
, we can speculate that HIF-1 and HIF-2 are upstream
and downstream regulators of RECK, respectively under
hypoxic conditions. But HIF-2 is directly involved in
RECKsilencing-mediated cell proliferation.
HIF-2 and c-Myc may collaborate to increase cell
proliferation under hypoxic conditions and in RECK silencing
cells. HIF-1 and HIF-2 have opposing effects on c-Myc
interaction and cell proliferation . Under hypoxic conditions,
HIF-1 and HIF-2 are competent to occupy c-Myc. HIF-1
participates in gene silencing at RECK promoter in accompany
with HDAC1 , and in the donating of c-Myc to HIF-2 rather
than binding to c-Myc to cause hypoxia-induced cell
proliferation. Since HIF-1 and -2 are expressed in a cell-type
specific manner , hypoxic conditions might have different
effects on cell proliferation depending on the availabilities of
HIF-1 and HIF-2 in a variety of cells. In a recent study,
HIF-2 was present in a stem cell population but HIF-1 was
present in both stem and non-stem cell populations and was
only stabilized under more severe hypoxic conditions .
HIF-2 transcriptionally activates the Oct4 gene, a stem cell
marker . A significant evidence supports our hypothesis
that HIF-2 alters the basic genetic activity of normal
transformed cells to a more stem-like hyperplastic phenotype in
a manner that depends on the fluctuating state of oxygen
availability under chronic pathophysiological conditions .
Furthermore, a switch of hypoxic response from HIF-1 to
HIF-2-depedent gene expression results in high aggressive
tumor through the promotion of stem cell characteristics . In
CD133+ glioblastoma cells, the expressions of MMPs and
RECK were induced by miR-125b, and the authors suggested
that the functional inhibition of RECK increases cancer stem
cell function . Our findings suggest that RECK is likely to be
an important molecule for regulation of switch from HIF-1- to
HIF-2- dependent transcription.
Although it has been shown that hypoxia induces cell cycle
arrest but not proliferation [31,32], exposure time and degree of
hypoxia can also affect cell cycle arrest/survival decisions. For
example, longer and more severe hypoxia induces cell cycle
arrest and apoptosis to a greater extent than shorter and milder
exposure . Hypoxia and the silencing of RECK by siRNA
Figure 6. Silencing of RECK increased in vitro soft-agar colony formation and in vivo tumorigenesis. (A) HEK293 cells
were transfected with siRECK and plated onto agar-coated 96-well plates 24 h after transfection. Colonies were observed one week
after seeding (upper panel). Original magnification 40X. Quantitation of colony formation by HEK293 cells transfected with
scrambled or RECK siRNA1 one week after seeding. Colony formation was measured using a fluorometer, as described in
Materials and Methods. Data presented as means SDs (n=4). *, p<0.01. (lower panel) (B) HEK293 cells stably transfected with
shRECK were injected subcutaneously into the flanks of nude mice. One week later, tumor volumes (length x width x height, mm)
were measured; animals were sacrificed 22 days after the first measurement. The upper panel shows tumors in sacrificed mice;
including the largest and smallest shRECK tumors (RECK) and the largest shlamin tumor (Lamin). RECK expression is shown
inside a graph with stable transfectant of shlamin and shRECK with RT-PCR. -actin was used as an internal control. Scale bar=10
mm. Data are presented as means SDs (n=4). *, p<0.01. Scale bar=100 m. (C) Immunohistochemical staining analysis with
antiCD31 (a, b), anti-phosphorylated-Rb (c, d), PCNA (e, f) and anti-p16 (g, h) antibodies in the control (shlamin) and shRECK tumors
shown in C. Anti-rhodamine (for CD31) and DAB (c-f) were used for visualization. Scale bar=10 m. (D) Immunohistochemical
staining analysis with anti-Hypoxic probe-1 (a, d), anti-CA9 (b, e), and anti-RECK (c, f) in xenofraft tumor section of stably
transfected shRECK or shlamin (control). Scale bar=100 m. (E) A scheme for the involvement of RECK/EGFR/HIF-2 in normal
epithelial proliferation and viability in hypoxia. A dotted line represents findings in reference 8.
increased the protein expressions of c-Myc, cyclin D1, cyclin A,
silencing involves a different pathway from involving
EGFRand p-pRb, but decreased those of p21cip1, p27kip1 and p16ink4A,
indicating that the G1/S transition is a key regulation point of
also involved in cell proliferation , MMP inhibition by RECK
the hypoxia-induced proliferation of epithelial cells. These
downregulation also suppresses cell proliferation induced by
results are consistent with the previous findings that RECK
hypoxia. These findings suggest that these two pathways work
depletion stimulates cell proliferation during successive cell
together to increase cell proliferation.
cultivations, and that this is correlated
expressions of p21cip1, p53, and p19ink4D in mouse MEFs .
Interestingly, it has been previously reported that the
In cancer cells, the repression of RECK by hypoxia also
macrophages results in the inductions of anti-apoptotic genes
influenced cell proliferation and enhanced migration or invasion
and cytokines that increase the survival and proliferation of
[21,23,33]. Yoshida et al. found that the inhibition of cancer cell
premalignant intestinal epithelial cells . In our findings,
proliferation is mediated by S-phase kinase-associated protein
2 (SKP2) targeting p27kip1 , which suggests that different
molecules might be involved in the suppression of cell growth
silencing may also participate the cell proliferation and survival.
by RECK in a cell-specific manner. Thus, the downregulation of
could perhaps be adopted as a means of preventing the early
transformation of hyperplastic cells in premalignant lesions
contribute to the development of the hyperplastic phenotype in
under hypoxic conditions. However, further investigations are
premalignant cells by increasing the expressions of genes
needed to characterize the
cancer cells increases the expression of EGFR  in
hyperplastic phenotype and during the cell cycle progression of
response to hypoxia . In a study using EGFR inhibitor, it
normal cells. The observation that hypoxia can cause RECK
silencing in normal cells is important, as it could be a novel
EGFR . However, our findings suggest that EGFR might be
mechanism for reducing tumor suppressor expression under
low oxygen tension-dependent stress, which characteristic of
and in cells under hypoxic conditions (Figure 4B). Importantly,
certain pathological conditions.
findings in VHL-/- cancer cells demonstrated that the
shRNAinduced spheroid formation in a three-dimensional tumorigenic
mediated tumorigenesis. Furthermore, the inactivation of EGFR
can be achieved by the restoration of RECK . Therefore, it is
determinant of the hyperplastic phenotype in hypoxic cells.
MMP inhibitor decreased proliferation rate
induced by hypoxia, MMP inhibition by hypoxia-induced RECK
Conceived and designed the experiments: YML. Performed the
experiments: MPN SHL KBL YML. Analyzed the data: SHL
YML MJK. Wrote the manuscript: YML GHP MJK. Critically
reviewed all the text: YML SHL MPN GHP MYL.
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