Pro-angiogenic effects of MDM2 through HIF-1a and NF

Mol Biol Rep (2014) 41:5533–5541
DOI 10.1007/s11033-014-3430-0
Pro-angiogenic effects of MDM2 through HIF-1a and NF-jB
mediated mechanisms in LNCaP prostate cancer cells
Praneetha Muthumani • Karthikeyan Alagarsamy •
Sivanesan Dhandayuthapani • Thiagarajan Venkatesan
Appu Rathinavelu
•
Received: 8 November 2013 / Accepted: 21 May 2014 / Published online: 28 June 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract Hypoxia stimulates several pathways that are
critical to cancer cell growth and survival, including activation
of vascular endothelial growth factor (VEGF) transcription.
Overexpression of VEGF and the extent of neoangiogenesis
are closely correlated with tumor development and cancer
metastases. Recent studies suggest MDM2 as one of the major
regulators of pro-angiogenic mechanisms. To assess the direct
correlation of HIF-1a and NF-jB, and the actual mechanism
of MDM2 involved in the control over VEGF transcription,
we exposed the LNCaP and LNCaP-MST cells (MDM2
transfected) to hypoxia. Our experiments confirm that MDM2
activation can lead to significant decrease in the levels of p53
in MDM2 transfected LNCaP-MST cells than the wild-type
LNCaP cells. The results further suggest that MDM2 can be a
strong regulator of both p53 dependent and independent
transcriptional activity. Similarly, an increased level of other
transcription factors such as HIF-1a, P300, STAT3, pAKT
and NF-jB was observed. As a point of convergence for many
oncogenic signaling pathways, STAT3 is constitutively activated at high frequency in a wide diversity of cancers. Our
results indicate that STAT3 can directly regulate VEGF
expression that is controlled by MDM2. Furthermore, it is
evident from our results that NF-jB may interfere with the
transcriptional activity of p53, by downregulating its levels.
On the other hand, several pro-angiogenic mechanisms,
including VEGF transcription which is controlled by MDM2,
seem to be mediated by NF-jB.
P. Muthumani K. Alagarsamy S. Dhandayuthapani T. Venkatesan A. Rathinavelu (&)
Rumbaugh Goodwin Institute for Cancer Research, College of
Pharmacy, Health Professions Division, Nova Southeastern
University, 1850 NW 69th Avenue, Suite #5, Plantation,
FL 33313, USA
e-mail: [email protected]
Keywords MDM2 Prostate cancer HIF-1a Proangiogenesis Hypoxia
Tumor angiogenesis is a process of formation of new
capillaries from an existing blood vessel, which is necessary for cancer cell survival and tumor growth. Angiogenesis that occurs during tumor growth is a regulated
process involving several growth stimulators such as vascular endothelial growth factor (VEGF) [1], fibroblast
growth factor (FGF), platelet derived growth factor
(PDGF) [2], angiopoietins [3], activators of integrins [4]
etc. In this process inhibitors such as thrombospondin [5],
angiostatin [6], and endostatin [7] also have important role.
Among these stimulatory and inhibitory factors, VEGF is
considered to be the most potent stimulator, which plays an
important role in promoting the growth, migration, and
assembly of endothelial cells during different stages of
angiogenesis [8–11]. Since VEGF transcription is up-regulated by hypoxia [12, 13], whenever cancer cells are
subjected to hypoxic environment they secrete VEGF,
which eventually binds to high affinity signaling receptors
on the endothelial cells of existing blood vessels. This
interaction of VEGF with its receptors subsequently leads
to the formation of new blood capillaries, which provide
the necessary nutrients and oxygen to the cancer cells via
newly established blood circulation. We have previously
reported the co-expression of VEGF in many cancer cell
lines which are positive for MDM2 expression [14].
MDM2 is a cellular oncoprotein encoded by a gene
located on chromosome 12q13-14 [15]. Amplification of
the MDM2 gene or over expression of the MDM2 protein
have been reported in 28 different types of human cancers,
including lymphoblastic leukemia (15), bronchogenic carcinoma (16), lymphoma (17) and bladder cancer (18).
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Furthermore, MDM2 over expression has been very well
correlated with aggressive tumor growth, lack of response
to chemoradiotherapy and poor survival [16–18]. Interestingly, amplification of MDM2 has been found in 35 % of
soft tissue sarcomas (11), one-third of malignant fibrous
histiocytomas (12), 42 % of liposarcomas (13), and 7 % of
osteosarcomas (14), and in all cases of salivary gland
carcinomas (19). In cancers, the multi-functional MDM2
protein typically inhibits the activity of p53 tumor suppressor protein either by masking its transactivation
domain or by directly binding and degrading p53 by
ubiquitination in the amino-terminal region and marking it
for proteosomal degradation through an E3 ubiquitin ligase
activity [15, 19–22]. Though MDM2 has been shown to
impact the cancer growth by many different mechanisms
involving p53 dependent and p53 independent pathways,
both in vivo and in vitro data obtained from some of the
laboratories so far have proved important for MDM2 in
controlling VEGF expression [23–25]. However, the
molecular mechanism involved in this process has not yet
been completely elucidated.
It is established that both HIF-1a and STAT3 can regulate VEGF expression and these transcription regulators
can be activated by oncogenes such as Src. However,
cooperative control of the VEGF promoter by each of these
factors has not been fully established. In regulating VEGF
transcription, both HIF-1a and STAT3 are shown to bind to
the transcriptional co-activator CBP/p300, suggesting that
if simultaneous occupancy of the VEGF promoter occurs,
they may be part of a single transcriptional complex [26].
In support of this putative mechanism, co-immunoprecipitation of HIF-1a and STAT3 along with CBP/p300 has
been reported in several studies [27]. Interestingly an
increased level of these two transcription factors during Src
activation has also been reported in PANC-1 and PC-3 cell
lines, which suggests that HIF-1a and STAT3 are components of a large complex governing transcription of
VEGF [27]. Thus, the co-activator p300 can bind to a
variety of transcription factors including STAT3 and HIF1a, and act as a critical component in hypoxia [26, 28].
Gene transcription by p300 is controlled in part by its
ability to bind upstream transcription factors, such as Ref1/APE in the composite VEGF regulatory element, and coordinate the transcription machinery.
The NF-jB family of transcription factors is another
major regulator of gene transcription that is primarily
involved in regulating immune, inflammatory and stress
responses. However, recently NF-jB was also shown to
have an important role in regulating tumor angiogenesis
[29, 30]. In addition, the role of NF-jBin controlling the
expression of VEGF has been reported in many cancer
cells. A direct correlation between inhibition of NF-jB and
reduced expression of VEGF has been reported in three
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Mol Biol Rep (2014) 41:5533–5541
different breast cancer cell lines, MCF-7, T47D, and MDAMB-231, and in all of them the basal levels of VEGF
mRNA expression was well correlated with those of
nuclear NF-jB activity. This has suggested the presence of
NF-jB binding site in the VEGF promoter region among
other promoters such as HRE, AP1, AP2 and SP1 [31, 32].
The main aim of the study presented here was to evaluate the actual mechanism involved in mediating the
MDM2 control over VEGF transcription during tumor
angiogenesis. For this reason we analyzed the levels of
transcription factors such as HIF-1a, P300, and STAT3 in
both wild type LNCaP cells and MDM2 transfected LNCaP
(LNCaP-MST) cell lines under both normoxic and hypoxic
conditions. In addition, we analyzed some of the up-stream
regulators of the PI3K/AKT pathway, including p53. The
results of our study are presented here and discussed in the
context of MDM2 mediated regulation of VEGF transcription through PI3K and NF-jB mediated pathways.
Materials and methods
Cell culture
The LNCaP prostate cancer cell line was a generous gift
from Dr. Thomas Powell (Cleveland Clinic Foundation,
Cleveland, OH). The LNCaP-MST (MDM2-transfected)
cells were kindly provided by Dr. Alan Pollack (Fox Chase
Cancer Center, Philadelphia, PA). The LNCaP and
LNCaP-MST cells were maintained in RPMI and Dulbecco’s modified Eagle’s medium (DMEM)-F12 [14]
containing 10 % fetal bovine serum (Hyclone, Logan, UT),
1 % L-glutamine, 1 % antibiotic–antimycotic solution (Life
Technologies, Gaithersburg, MD) respectively. The cancer
cells were grown in a humidified air/CO2 (19:1) atmosphere at 37 °C and replenished with the respective growth
media before each experiment.
Hypoxia stimulation
For all hypoxia experiments, the cells were grown to
80–90 % confluency and, on the day of experiments, they
were replenished with the respective growth medium
containing 2 % FBS. Culture dishes were then placed in
airtight modular incubator chamber (Billups-Rothenberg,
Inc., Del Mar, CA) that was saturated with pre-analyzed
gas mixture 1 % O2, 5 % CO2, 94 % N2 (Praxair Inc.,
Miami, FL) and humidity. This incubator was kept at 37 °C
for 12 h. The normoxic cells were placed at 37 °C in a
humidified air/CO2 (19:1) atmosphere. Both hypoxia and
normoxia exposed cells were always assayed at the same
time.
Mol Biol Rep (2014) 41:5533–5541
Western blot analysis
At the end of 12 h incubation period, both the normoxic
and hypoxic cells were lysed by sonication and the cell
lysates were subjected to western blot experiments
according to the modified method of Rathinavelu et al.
[33]. Exactly 30 lg of protein from each sample was
resolved on 7.5 % SDS-polyacrylamide gel. The proteins
were then transferred onto the nitrocellulose membrane and
probed with 1:200 dilution of anti-MDM2 monoclonal
antibody (Ab-1) (Santa Cruz Biotechnologies, Santa Cruz,
CA). The immunoreactive MDM2 protein signals were
detected using ECL blot-developing system (Amersham
Corporation, Piscataway, NJ).
For the detection of p53, VEGF, HIF-1a, P300, STAT3,
and NF-jB protein levels, 30 lg aliquots of the protein
samples were subjected to electrophoresis on 7.5 % polyacrylamide gel and then they were transferred onto the
nitrocellulose membrane. After blocking with 5 % non-fat
dry milk solution or 5 % BSA as per manufacturer’s recommendation, the membranes were probed with (1:500
dilution) anti-HIF-1a monoclonal antibody (BD Transduction Laboratories, CA), anti-VEGF, p53, P300 antibodies (Santa Cruz Biotechnologies, CA) NF-jB and
STAT3 (cell Signaling, MA). The MDM2, p53, VEGF,
HIF-1a, p300 and NF-jB protein bands were visualized
using Amersham chemiluminescence kit after incubation
of the blotted membrane with HRP conjugated secondary
antibody (Amersham, Piscataway, NJ). As a loading control, b-actin or GAPDH western blots were developed
using a 1:2,000 dilution of specific antibodies (Sigma, St.
Louis, MO), and using the same protein samples.
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protein when compared to non-transfected LNCaP cells
(P \ 0.05) (Fig. 1a). Subsequently, in order to determine
the influence of MDM2 on VEGF protein expression, we
analyzed the levels of VEGF in both LNCaP and LNCaPMST cell lines. We observed about 88 % more expression
of VEGF in MDM2 transfected LNCaP-MST cells compared to non-transfected LNCaP cells (P \ 0.05) (Fig. 1b).
Furthermore, we were interested in examining the levels of
HIF-1a in the cells where VEGF expression was enhanced
due to MDM2 transfection. The results showed HIF-1a
protein expression in both the wild type and MDM2
transfected prostate cancer cell lines under hypoxic and
normoxic conditions. We observed 97 % higher expression
of HIF-1a in LNCaP-MST cells compared to LNCaP cells
(P \ 0.05) (Fig. 1c).
Down regulation of p53 in MDM2 transfected cells
under both normoxic and hypoxic conditions
The role of p53 in inducing cell cycle arrest and apoptosis
is a well established mechanism in many cells. Therefore,
to study the effect of MDM2 overexpression on the levels
of p53 in LNCaP cells, we analyzed the expression status
of these genes under both normoxic and hypoxic conditions. The p53 level in LNCaP-MST cells were only 28 %
over the non-transfected cell levels under normoxic condition, whereas under hypoxic condition, it was reduced
even further to about 16 % (P \ 0.05) (Fig. 2a, b). In
addition to finding a noticeable decrease in the levels of
p53 in the whole cell lysate, there was significant reduction
in the levels of p53 in nuclear fraction (Fig. 2b).
Statistical analysis
Levels of PI3K and pAKT in MDM2 transfected cells
The results were expressed as mean ± SD. The statistical
significance between groups analyzed by one-way analysis
of variance (ANOVA) followed by Student–Newman–
Keuls multiple comparisons tests. The P values \ 0.05
were considered significant and presented in the results.
Results
The cell lysates from normoxic and hypoxic cells were
analyzed for PI3K and pAKT levels in both LNCaP and
LNCaP-MST cells. As shown in Fig. 3a, the PI3K level
was 96 % higher in MDM2 transfected LNCaP-MST cells
during hypoxia compared to the non-transfected LNCaP
cells (P \ 0.05). This increase in the PI3K levels seems to
have resulted in producing higher levels of pAKT as presented in Fig. 3b.
Expression of VEGF, and HIF-1a in MDM2 transfected
cells
Expression of NF-jB under both normoxic and hypoxic
conditions in MDM2 transfected cells
Typically the wild type LNCaP cells express detectable
levels of MDM2. In addition to already elevated levels of
MDM2, when LNCaP cells were transfected stably with
MDM2 gene expressing plasmid (neomycin-selectable
pCMV-MDM2 expression plasmid), the transfected
LNCaP-MST cells expressed about 109 % more MDM2
The role of NF-jB in tumor angiogenesis has been recognized for some time. In this study, we have observed that
the levels of transcriptional factor NF-jB level was
increased significantly in MDM2 transfected in LNCaPMST cells during hypoxia and that increase was 43 %
compared to non-transfected cells (P \ 0.05) (Fig. 3c).
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Mol Biol Rep (2014) 41:5533–5541
Fig. 1 Levels of MDM2,
VEGF and HIF-1a protein in
LNCaP and LNCaP-MST cells.
Western blot analysis of whole
cell lysates was carried out to
assess a MDM2, b VEGF and
c HIF-1a levels using specific
antibodies to MDM2, VEGF
and HIF-1a in wild-type LNCaP
and MDM2 transfected LNCaPMST cells. As a loading control
GAPDH blots were developed
using specific antibodies with
the same protein samples. The
densitometry scanning data
from the blots of at least three
experiments are given as
mean ± SD and the level of
statistical significance is
indicated by *(P \ 0.05) in
comparison to respective
controls
Fig. 2 Down regulation of p53 in MDM2 transfected cells. Western
blot analysis shows the level of p53 protein in a whole cell extract,
b cytoplasmic extract and c nuclear extract of LNCaP and LNCaPMST cells after 12 h exposure to hypoxia. The p53 target protein
level was greatly reduced in nuclear extract upon 12 h exposure to
hypoxia. Immunoblots were developed using same protein samples
with specific antibody to b-actin as a loading control
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Recent studies have revealed that in general STAT3 can
also be involved in the control of VEGF transcription in
prostate cancer cells. In this study the levels of STAT3
were analyzed under both normoxic and hypoxic conditions to further establish the role of this gene in MDM2
transfected LNCaP-MST cells, where VEGF expression is
significantly elevated. As shown in Fig. 4a, the expression
of STAT3 was 35 % higher in LNCaP-MST than in
LNCaP cells (P \ 0.05). These results suggested that
STAT3 may also have an important role in elevating VEGF
transcription in MDM2 transfected prostate cancer cells
that were tested in our experiments. Hence, to fully
understand the effects of MDM2 on the levels of p300, we
studied the expression of p300 also in LNCaP and MDM2
transfected LNCaP-MST cells. In LNCaP-MST cells, p300
expression level was elevated by almost four folds under
normoxic condition, compared to LNCaP cells (P \ 0.05)
(Fig. 4b).
Mol Biol Rep (2014) 41:5533–5541
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Fig. 3 Ectopic overexpression
of MDM2 increases PI3K,
pAKT and NF-jB levels.
Western blot analysis of whole
cell lysates was carried out to
assess a PI3K, b pAKT and
c NF-jB levels in both LNCaP
and LNCaP-MST cells after
12 h exposure to hypoxia.
Immunoblotting was probed
with antibodies specific to
human PI3K, pAKT and NFjB. The levels of PI3K, pAKT
and NF-jB expression were
significantly higher in MDM2
transfected LNCaP-MST cells
compared to the non-transfected
cells. The bar graphs next to the
blot show the relative change in
expression levels. The error
bars in a, b, and c represent the
SD from at least three
experiments and the level of
statistical significance is
indicated by *(P \ 0.05) in
comparison to respective
controls
Fig. 4 Activation of STAT3
and p300 in MDM2 transfected
cells. Western blot analysis
using specific antibodies to
STAT3 and p300 shows the
levels of a STAT3 and b p300
in LNCaP and LNCaP-MST cell
lysates. The bar graphs next to
the blot show the change in
expression. The error bars in
a and b represent the SD from
experiments performed in
triplicate and the level of
statistical significance is
indicated by *(P \ 0.05) in
comparison to respective
controls
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Discussion
It has been very well established in the last two decades
that tumor angiogenesis plays an important role in cancer
growth and metastasis. VEGF is one of the most important
factors involved in regulating the process of angiogenesis.
As indicated earlier, angiogenesis could be regulated by
multiple mechanisms that are already known. However,
some recent studies have indicated that MDM2 can also
play an important role in controlling angiogenesis through
regulation of VEGF expression [14, 34–37]. The exact
pathway linking MDM2 to VEGF expression has not yet
been identified. Our current studies using LNCaP and
MDM2 transfected LNCaP (LNCaP-MST) prostate cancer
cells have revealed one of the pathways that may be
involved in propagating MDM2-mediated control over
VEGF expression. As shown in the results, the LNCaPMST cells expressed significant higher levels of VEGF
compared to non-transfected LNCaP cells which coincides
with the increase in the levels of several components of the
pro-angiogenic pathways. Activation of PI3K-mTOR
pathway was reported in several instances when there was
an increase in the expression of VEGF in both normoxic
and hypoxic conditions [38]. This is in conformity with the
present result. Since PI3K-mTOR pathway requires HIF-1a
as the primary member of the transcriptional complex, it is
generally believed that activation of this pathway works
more effectively under hypoxic conditions for inducing
VEGF transcription. However, from our current results it
appears that in MDM2 transfected cells, not only the PI3KmTOR pathway might be very active but also the basal
level of HIF-1a is high [39]. This confirmed our speculation that MDM2 transfection may have significantly
increased the levels of VEGF expression via enhancing the
expression of HIF-1a level in LNCaP and LNCaP-MST
prostate cancer cells. This analysis yielded very interesting
results in support of our speculation about activation of the
PI3K-mTOR pathway towards stimulation of the VEGF
transcription. Though elevation of HIF-1a levels during
normoxic conditions was considered as an unlikely possibility, it has been shown that the HIF-1a levels can be
increased in normoxic conditions following doxorubicin
treatment in MCF-7 cancer cell line [40]. Similarly in the
present studies, MDM2 appears to be causing the elevation
of HIF-1a under normoxic conditions concurrent with
increasing expression of VEGF in these normoxic cells
even in the absence of hypoxic stress. In support of this
observation some of the recent studies have indicated a
possible role for HIF-1a in controlling VEGF transcription
in MDM2 expressing cells [32, 33]. Some of the earlier
reports have further indicated that activation of PI3KmTOR pathway is one of the reasons for the elevation of
transcription factor NF-jB [41]. In addition to its role in
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controlling the expression of pro-angiogenic factors such as
VEGF, NF-jB has been shown to regulate the expression
of several cytokines that can cause inflammatory responses
in cancer [42]. More importantly, a functional antagonism
between NF-jB and p53 has been reported recently where
they have also reported a reciprocal regulation between the
levels of these factors [43]. In addition to the PI3K-mTOR
pathway, it appears that STAT3-mediated mechanisms also
seem to be enhanced subsequent to MDM2 transfection in
LNCaP-MST cells.
Consistent with our speculation, the present data indicated that STAT3 should have an important role in MDM2
mediated activation of VEGF transcription because, its
levels were found to be elevated in MDM2 transfected
LNCaP-MST cells compared to the non-transfected cells.
Our laboratory has previously reported the elevation of
pSTAT3, in addition to increased levels of HIF-1a, in
MDM2 transfected LNCaP-MST cells [44]. The present
observation is reaffirmation of our earlier reports, further
supported by a significant decrease in the levels of p53 in
both the whole cell lysate and nuclear fraction of MDM2
transfected LNCaP-MST cells. The p53 tumor suppressor
genes is not only required for maintaining normal cell
function, it is also necessary for elimination of mutated
genes and dead cells, by such cellular processes as cell
cycle progression, apoptosis and cellular differentiation.
p53 is one of the most important tumor suppressor genes
responsible for the regulation of angiogenesis [45]. Furthermore, p53 serves as an important nuclear transcription
factor, with ability to transcriptionally activate target genes
such as p21, GADD45, Bax, Puma, and Noxa. Expressions
of these genes are necessary to induce G1 cell cycle growth
arrest and apoptosis [45, 46], in response to cellular stress,
including DNA damage and oncogene activation. On the
other hand when expressed in high levels, MDM2 can
interact with p53 to inhibit p53’s transcriptional activity
and stop many of its tumor suppressor functions [47].
Furthermore, some of the recent evidences clearly show
that p300, which is known to be an important transcription
co-activators in the pro-angiogenic pathway, seems to play
a significant role in controlling VEGF expression by supporting the transcription function of HIF-1a. Sometimes,
MDM2 can interfere with the ability of p53 to contact
transcriptional co-activators such as p300/CBP also [47]
and as a result the cell cycle control and anti-angiogenic
mechanisms are lost. Thus, our results clearly suggest that,
MDM2 transfection could significantly impact the expression of several genes including p300 and NF-jB that might
eventually causes higher level expression of VEGF due to
the interconnected mechanisms. In addition to direct
binding to p53, MDM2 also promotes ubiquitination of p53
and degrades the latter through lysosomal export mechanism [48–50]. Thus, in the results presented here it can be
Mol Biol Rep (2014) 41:5533–5541
inferred that, MDM2 transfection significantly reduces the
levels of p53 even under normoxic conditions, while under
hypoxia, the p53 protein level were reduced lot more
favoring an increased VEGF transcription.
It appears that HIF-1a mediated transcriptional control is
crucial in the mechanisms that are influenced by MDM2 [34,
51, 52]. This speculation is greatly substantiated by the
increases in the levels of HIF-1a, p300 and NF-jB found in
MDM2 transfected LNCaP-MST cells. The transcription
factor NF-jB, is frequently detected in tumors [53] and it has
been shown to regulate multiple anti-apoptotic genes and to
enhance tumor growth [54]. In order to explain its role in cell
cycle control and possibly in pro-angiogenic pathway, [55]
some of the previous studies have reported that NF-jB can
induce the expression of MDM2 by binding with its P1
promoter region. In this respect, different molecular mechanisms of NF-jB activation have been proposed that will
eventually support tumor progression [56, 57]. For example,
Ying et al. [58] reported that loss of PTEN can promote NFjB activity. In a reciprocal way three of the recent reports
have also shown that NF-jB can down regulate the expression of PTEN [59–61]. Furthermore, NF-jB has been shown
to interfere with the transcriptional activity of p53 and induce
the expression of VEGF. Thus, critical role of NF-jB and
STAT3 are still evolving as far as cancer growth and angiogenesis is concerned. Various factors that are involved in
the control of the transcriptional activation of NF-jB, such as
TNF, bacterial infection, viral infection, DNA damaging
agents and other forms of cellular stress [62, 63] and ARF
(known as 14ARF in humans and P19ARF in mouse) work
through p53 dependent pathway. The present result further
substantiate the mechanism exerted by MDM2 that could
work via p53 dependent or p53 independent pathways to
increase NF-jB levels [64–66]. However, the exact mechanism involved in activating the NF-jB during MDM2
mediated VEGF transcription control requires further scrutiny in order to have better understanding.
In conclusion, so far our results have clearly indicate
that, MDM2 plays the role of master regulator in inducing
VEGF transcription by suppressing p53 and significantly
increasing the levels of important genes such as STAT3,
NF-jB, HIF-1a, and p300. Further studies in this direction
will shed more light on the in-depth mechanisms involved
in driving this pro-angiogenic machinery.
Acknowledgments We would like to thank Dr. Thomas Powell
(Cleveland Clinic Foundation, Cleveland, OH, USA) and Dr. Alan
Pollack (Fox Chase Cancer Center, Philadelphia, PA, USA) for their
kind gift of LNCaP and LNCaP-MST cells, respectively. We
acknowledge the Royal Dames of Cancer Research at Fort Lauderdale,
Florida and President’s Faculty Research and Development Grant
(PFRDG) of Nova Southeastern University (NSU) for their support.
Conflict of interest
The authors report no conflicts of interest.
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