NF-κB potentiates tumor growth by suppressing a novel target LPTS
Liu et al. Cell Communication and Signaling
NF-κB potentiates tumor growth by suppressing a novel target LPTS
Dongbo Liu 0 1
Hongping Miao 0 2
Yuanyin Zhao 0 1
Xia Kang 1
Shenglan Shang 1
Wei Xiang 1
Rongchen Shi 1
Along Hou 1
Rui Wang 1
Kun Zhao 1
Yingzhe Liu 1
Yue Ma 1
Huan Luo 1
Hongming Miao 1
Fengtian He 1
0 Equal contributors
1 Department of Biochemistry and Molecular Biology, Third Military Medical University , Chongqing 400038 , China
2 Department of Neurosurgery, Southwest Hospital, Third Military Medical University , Chongqing 400038 , China
Background: Chronic inflammation is causally linked to the carcinogenesis and progression of most solid tumors. LPTS is a well-identified tumor suppressor by inhibiting telomerase activity and cancer cell growth. However, whether and how LPTS is regulated by inflammation signaling is still incompletely elucidated. Methods: Real-time PCR and western blotting were used to determine the expression of p65 and LPTS. Reporter gene assay, electrophoretic mobility shift assay and chromatin immunoprecipitation were performed to decipher the regulatory mechanism between p65 and LPTS. Cell counting kit-8 assays and xenograt models were used to detect p65-LPTSregulated cancer cell growth in vitro and in vivo, respectively. Results: Here we for the first time demonstrated that NF-κB could inhibit LPTS expression in the mRNA and protein levels in multiple cancer cells (e.g. cervical cancer and colon cancer cells). Mechanistically, NF-κB p65 could bind to two consensus response elements locating at −1143/−1136 and −888/−881 in the promoter region of human LPTS gene according to EMSA and ChIP assays. Mutation of those two binding sites rescued p65-suppressed LPTS promoter activity. Functionally, NF-κB regulated LPTS-dependent cell growth of cervical and colon cancers in vitro and in xenograft models. In translation studies, we verified that increased p65 expression was associated with decreased LPTS level in multiple solid cancers. Conclusions: Taken together, we revealed that NF-κB p65 potentiated tumor growth via suppressing a novel target LPTS. Modulation of NF-κB-LPTS axis represented a potential strategy for treatment of those inflammation-associated malignancies.
NF-κB; LPTS; Promoter; Cervical cancer; Colon cancer
Chronic inflammation is causally linked to the
carcinogenesis and development of most solid tumors [
The proinflammatory signal in cancers is characterized
by the activated inflammatory pathways (e.g. NF-κB and
JNK), elevated inflammatory cytokines (e.g. IL-1β, IL-6
and TNFα) and increased infiltration of immune cells
(e.g. macrophages and lymphocytes) [
]. In fact, the
inflammatory microenvironment caused by microbial
infection plays a very important role in malignant
transformation and cancer progression. For example, cervix
infection by HPV16 is an independent and high risk
factor of cervical cancer [
pylorimediated chronic atrophic gastritis is suggested to be a
precancerosis of gastric cancer [
]. Similarly, imbalance
of intestinal microbiota is also causally linked to
intestine inflammation and tumors [
It has been revealed that NF-κB is a potent contributor
in cancer progression by enhancing the expression of
oncogenes and activation of onco-signals [
]. As a
transcription factor, NF-κB controls the transcription
specificity via the assembly of heterodimers or homodimers of
five different NF-κB proteins (p65, p50, c-Rel, p105 and
]. In response to proinflammatory stimulation,
the I-κB kinase is activated to phosphorylate I-κB
protein and suppress I-κB-mediated p65 degradation [
P65 contains a DNA binding domain and mediates the
transcription function of NF-κB in most situations [
LPTS, also named PINX1, is a well-characterized
tumor suppressor by inhibiting telomerase activity in
multiple cancers [
]. It has been reported that LPTS
expression is deficient in multiple cancers and positively
correlated to prognosis [
]. Mechanistically, LPTS
inhibited tumor cell growth by directly suppressing the
activity of human telomerase reverse transcriptase
(hTERT) or reducing c-myc-mediated hTERT
]. However, how the expression of LPTS is
repressed in a special cancer type is still incompletely
Modulation of inflammatory signaling is associated
with alteration of some important regulators in cancer
progression (e.g. c-myc and p53) [
]. We previously
reported that HPV16 E6 could suppress p53-dependent
LPTS expression in cervical cancer cells [
that HPV16 E6 is also a proinflammatory stimulator
], we aimed to investigate whether inflammatory
signals would regulate LPTS expression in
inflammationassociated cancer cells (e.g. cervical cancer and colon
cancer). We would also investigate the regulatory
mechanism of LPTS in vitro and in xenograft models.
CaSki cell, a HPV16/HPV18-positive cervical carcinoma
cell line, and MKN-45 cell, a human gastric carcinoma
cell line, were purchased from American Type Culture
Collection (Rockville, MD, USA). MC-38, a mouse
colorectal cancer cell line, was maintained in our lab [
All the cells had been authenticated and tested for
mycoplasma. Those cells were grown in high glucose
DMEM supplemented with 10% fetal bovine serum
(FBS), penicillin G (100 units/ml) and streptomycin
(100 μg/ml) at 37 C° in a humidified 5% CO2
atmosphere. Cells at approximately 80% confluence were
washed with PBS and preincubated in serum-free
medium for 2 h before treatment with the NF-κB
inhibitor BAY11–7082 (S1523, Beyotime, Shanghai, China).
All the mouse experiments were approved by the
Institutional Animal Care and Use Committee of the Third
Military Medical University and carried out in
accordance with the "Guide for the care and use of laboratory
animals" published by the US National Institutes of
Health (Publication no.85–23, revised 1996). All the
mice were purchased from the Institute of Experimental
Animal in Third Military Medical University
(Chongqing, China). Each group of mice was maintained five
per cage supplied with a regular rodent diet and
standard water ad libitum in a pathogen-free facility with a
12-h light, 12-h dark cycle.
Subcutaneous xenograft models
Eight weeks old female BALB/c nude mice were
subcutaneously inoculated with CaSki cells or MKN-45 cells
(5.0 × 106/100 μl PBS) in the groin. 6–8 weeks old
female C57BL/6 mice were subcutaneously injected with
MC-38 cells (5.0 × 106/100 μl PBS) in the groin. All the
inoculated cancer cells were stably transfected with
shRNAs of LPTS or p65. For overexpression of E6, the
CaSki cells were transfected transiently. The NF-κB
inhibitor BAY11–7082 was injected intraperitoneally at
5 mg/kg dissolved in DMSO/PBS buffer thrice per week.
The control group received the equal volume of vehicle
only. The volume of the tumor was determined
dynamically as described previously [
]. The survival time of
those mice were also recorded.
Transfection was performed according to the protocol of
Lipofectamin-2000 (#11668019, Invitrogen, Shanghai,
China). Briefly, cells were plated 24 h before transfection
at a density of 1.0 × 105/well on a 24-well plate
(#CLS3524, Sigma, Shanghai, China). CaSki or MC-38
cells were incubated 4 h before transfection with FBS-free,
antibiotic-free media and then transfected with plasmids
(0.2 μg/ml) or shRNAs (20 nmol/ml). After transfection
for 6 h, the medium was removed and replaced with
complete growth medium for further treatment. For
overexpression of HPV16-E6 in the CaSki cells, the plasmid
pEGFP-HPV16-E6 or the control vector pEGFP-N1 was
constructed and transiently transfected respectively, as
described in our previous report [
Packaging and transfection of lentiviruses
P65 and LPTS shRNA constructs for lentivirus
packaging were purchased from Sigma. The sequences used
for human p65 silence were: 5′- CCGGAGAGGACATT
CTCTTTTTTG-3′ (p65-shRNA-1); 5′- CCGGCCCTG
TGCTCAGGGTTTTTG-3′ (p65-shRNA-2). The
sequences used for human LPTS silence were: 5′- CCGGGA
ACCTGCGTCTCTTTTTG -3′ (hLPTS-KD). The
sequences used for mouse p65 silence were: 5′- CCGGCCC
GTGCTGAGGGTTTTTG -3′ (mp65-KD). The sequences
used for mouse LPTS silence were: 5′- CCGGCCGGGTT
ACCCGGTTTTTG-3′ (mLPTS-KD). The control shRNA
plasmid was also provided by Sigma (#SHC016-1EA). The
Caski cells and MC-38 cells with stable silence of p65,
LPTS or both were constructed for the assays of cell
growth in vitro and in vivo.
Reporter gene constructs and assays
The reporter gene pGL3-C1harboring the promoter
region (−2303/+42) of human CCL20 was used to indicate
the transcription activity of NF-κB, as a NF-κB binding
element (GGGGAAAACCCC) locating at −81/−70 was
identified in our previous study [
]. The pGL3-basic
vector-based reporter gene containing the human LPTS
promoter region −1300/+25 or −495/+25 was
constructed. Two potential NF-κB binding sites locating at
−1143/−1136 (tgggaaaa) and −888/−881 (tggagagt) in the
human LPTS promoter region were mutated to
MUT1(tgtctaaa) and MUT-2 (tgcatagt) respectively or
simultaneously (MUT-3) in the reporter constructs according
to a site-directed mutation protocol (#D401, TaKaRa
MutanBEST Kit, TaKara, Japan) as described previously
] (See in Fig. 3c). The mutation primers were
designed as follows: for MUT-1, the forward:
5′ttggctgtctaaattccattcact-3′, the reverse:
5′-ggcaggaaagctgtgacattgtga-3′; for MUT-2, the forward:
5′ttactgcatagtcactcacccaa-3′, the reverse:
5′-acttcaggtgacagtgcacaca-3′. Each reporter construct was transfected
into the CaSki cells for more than 24 h. The cells were
washed twice with PBS and lysed with specific reporter
lysis buffer. Then, the luciferase activities of the cell
lysate were evaluated according to the manufacturer’s
instructions (#E1910, Promega, Shanghai, China), and the
total protein concentration in each well was measured
as an internal control. The final results were displayed as
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated using TRIzol reagent
(Invitrogen; Thermo Fisher Scientific, Inc.). 1 μg RNA was
reverse transcribed into cDNA using the RevertAid First
Strand cDNA Synthesis kit (#K1622, Thermo Fisher
Scientific, Inc.) according to the manufacturer’s protocol.
qPCR was carried out using a ABI 7500 Real-Time PCR
system (Applied Biosystems; Thermo Fisher Scientific,
Inc.). The mRNA expression levels were normalized to
β-actin. Reactions were performed in duplicate using a
SYBR kit (TakaRa, Shiga, Japan) The primers were
designed and synthesized upon request. The amplification
steps consisted of denaturation at 95 °C, followed by
40 cycles of denaturation at 95 °C for 15 s and then
annealing at 60 °C for 1 min. Relative target gene
expression was calculated using the 2-ΔΔCq method [
Cells were lysed with RIPA Lysis and Extraction Buffer
(#P0013C, Beyotime, Shanghai, China) supplemented
with 1% protease inhibitor cocktail (#P8340, Sigma), as
well as 1 mM phosphatase inhibitors (#2850, Sigma) and
shaken for 30 min before centrifugation at 12000 g for
30 min at 4 °C. The supernatant was collected and
quantified using a BCA kit (#P0009, Beyotime, Shanghai,
China). The extracted proteins (50 μg/well) were
separated through SDS-PAGE on a 10% gel, and transferred
to a polyvinylidene difluoride membrane. The membrane
was blocked with 5% non-fat milk at 4 °C overnight, and
then incubated with anti-LPTS (#H00054984-K,
Abnova), anti-p65 (#8242, Cell signaling) and
anti-βactin (#3700, Cell signaling) for 10 h at 4 °C. The
membrane was rinsed 3 times with PBS containing 0.1%
Tween 20 (PBST), and incubated with the appropriate
horseradish peroxidase-conjugated second antibody for
1 h at room temperature. The membrane was then
washed with PBST for 3 times and incubated with
enhanced chemiluminescence substrate (#NEL105001EA,
PerkinElmer) for 1 min at room temperature. The
signals were captured using a ChemiDoc Touch™ Imaging
system (Bio-Rad Laboratories).
Electrophoretic mobility shift assay (EMSA)
The nuclear extracts were prepared from the empty vector
pEGFP-N1 or pEGFP-HPV16-E6 transfected -CaSki cells.
EMSA was performed according to the manufacturer’s
instruction (#GS002, Beyotime, Shanghai, China). The
biotin-labeled probes harboring the potential NF-κB
binding elements (−1150/−1125, 5′-CCTTGGCTGGGAAAAT
TCCATTCACT-3′; −895/−870, 5′- AGTTTACTGGAGA
GTCACTCACCCAA -3′) were designed and synthesized
(Sbsgene, Shanghai, China). The underlined bases indicated
the core sequences.
Chromatin immunoprecipitation (ChIP)
The binding activity between p65 protein and the
potential NF-κB binding elements (−1143/−1136,
5′TGGGAAAA-3′; −888/−881, 5′-TGGAGAGT-3′) in the
promoter region of human LPTS in CaSki cells was
measured using the ChIP test. Briefly, CaSki cells were
fixed with 1% of formaldehyde and then lysed in cell
lysis buffer (5 mM PIPES, 85 mM KCl, and 0.5% NP-40,
supplemented with protease inhibitors, pH 8.0) using a
dounce homogenizer to isolate the nuclei. The nuclei
were resuspended in nuclei lysis buffer (50 mM
TrisHCl, 20 mM EDTA, and 1% SDS, supplemented with
protease inhibitors, pH 8.1) and sonicated to shear
genomic DNA to an average fragment length of 200–
1000 bp. Lysates were centrifuged, and the supernatants
were collected. 50 μl of each samples was used as the
input control. The supernatants underwent overnight
immunoprecipitation (with IgG or p65), elution, reverse
cross-lining and protease K digestion according to
manufacturer’s protocol (#P2078, Beyotime, Shanghai,
China). Purified DNA extracts were analyzed by realtime
PCR using the primer pairs that cover the predicted
NFκB binding elements in the LPTS promoter region. The
primers for element (−1143/−1136) were 5′-CCTTCCT
GAGTCCAGTGC-3′ (forward) and 5′-GGGAGGCAAGT
GAATGGAA-3′ (reverse). The primers for element (−888/
−881) were 5′-CTGAAGTTTACTGGAGAG-3′ (forward)
and 5′- CAGGGGAGTTCTAATAAG-3′ (reverse). The
primers for input control were 5′-TTCACTTGCCTCCCC
TCAC-3′ (forward) and 5′-ACTCAGGTGCCAAGAA
Cell counting kit-8 (CCK8) assays
The cell viability of CaSki and MC-38 cells were
measured by CCK8 assays. The cells were seeded at
1.0 × 104 cells/well in 96-well plates. After overnight
incubation at 37 C° in a humidified 5% CO2 atmosphere,
the medium was removed and replaced with100 μl of
PBS containing 20 μl of CCK8 solution (5 g/l, Sigma,
USA). Plates were then incubated at 37 C° for 3 h before
measurement (Time point 0) using a microculture plate
reader at a wavelength of 450 nm. Then, the testing
solution was removed and the cells were subjected to
further treatment of NF-κB inhibitor (10 μM) or DMSO as
control. CCK8 assays were carried out at time point 24
and 48 h. The cell growth curve was obtained according
to the readout at the wavelength of 450 nm at different
Statistical analysis was performed using GraphPad Prism
version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA).
The results were expressed as the mean ± standard error
of the mean and were analyzed using a two-tailed unpaired
Student’s t-test or one-way ANOVA analysis of variance
followed by post hoc test for multiple comparisons.
P < 0.05 indicated a statistically significant difference.
Expression of LPTS is generally reduced in cancer tissues
It’s reported that LPTS is a tumor suppressor in multiple
]. From an open database (http://mer
av.wi.mit.edu/SearchByGenes.html), we demonstrated
that the mRNA level of LPTS was generally decreased in
cancer tissues of colon, female reproductive system,
kidney, liver, lung, pancreas and stomach, although the
LPTS expression is increased in breast and prostate
cancers (Fig. 1a -b). It’ reported that most of those cancers
underwent an inflammatory process before malignant
]. This clue indicated a potential
correlation between inflammatory signaling pathways
and LPTS expression.
NF-κB suppresses LPTS expression in mRNA and protein
The classic inflammatory pathways include NF-κB and
]. To investigate whether inflammatory signaling
would regulate LTPS expression, a
HPV16/HPV18-positive cervical carcinoma cell line (CaSki cell) was
employed, as NF-κB was activated in response to virus
]. We demonstrated that the NF-κB
inhibitor markedly suppressed NF-κB activity (Fig. 2a) and
potentiated LPTS expression in mRNA and protein levels
in CaSki cells (Fig. 2b-c). However, the JNK inhibitor
didn’t affect the expression of LPTS significantly (Data
not shown). For confirmation, we further silenced p65
expression and reduced NF-κB activity with p65-specific
shRNAs (Fig. 2d-e). Obviously, p65 shRNA-2 was more
efficient than shRNA-1. As expected, p65 shRNAs
markedly increased the expression of LPTS in mRNA and
protein levels (Fig. 2f-g).
NF-κB suppresses LPTS expression through cis-regulatory
Provided that NF-κB is a transcription factor and that
two putative NF-κB binding elements locating at −1143/
−1136 and −888/−881 in the promoter region of human
LPTS gene were predicted according to an
onlinesoftware (http://alggen.lsi.upc.es/), we next deciphered
whether NF-κB would regulate LPTS expression in the
promoter level. We constructed two reporter plasmids:
one harboring the promoter region −1300/+25
containing both potential NF-κB binding sites, the other
harboring the region −495/+25 without any predicted NF-κB
binding elements (Fig. 3a). Those reporter plasmids were
transfected into CaSki cells and luciferase activity was
determined. We demonstrated that the reporter gene
activity of −495/+25 region was notably higher than that of
the −1300/+25 region (Fig. 3b), indicating that those two
predicted binding sites of NF-κB might be functional in
regulating LPTS promoter activity. Thus, we next
performed site-directed mutation of those two binding
sites respectively or simultaneously, and further
evaluated their effects on LPTS promoter activity (Fig. 3c).
We revealed that simultaneous mutation of those two
sites greatly increased the reporter gene activity and the
proximal site (−888/−881) was more profound than the
distal one (−1143/−1136) (Fig. 3d). Especially,
inactivation of NF-κB with a specific inhibitor or p65 shRNA
markedly stimulated promoter activity of LPTS, while
simultaneous mutation of both NF-κB binding elements
fully abrogated this effect (Fig. 3e-f ).
NF-κB (p65) directly binds to LPTS promoter
To verify the direct binding between NF-κB (p65) and
its potential binding sites in LPTS promoter, EMSA and
ChIP assays were performed. In vitro, we demonstrated
that probe 1 harboring the proximal site (−888/−881)
incorporated more p65 proteins than probe 2 containing
the distal site (−1143/−1136) (Fig. 4a). In vivo, the
binding activity between p65 proteins and the predicted
binding elements were confirmed by ChIP assays.
Consistent with the EMSA test, we revealed that the binding
activity of p65 proteins to the proximal site was more
profound than the distal one (Fig. 4b-c).
HPV16-E6 promotes NF-κB-dependent growth of CaSki
Aforementioned results revealed the inhibitory role of
NF-κB in LPTS expression. However, the function of
NF-κB-LPTS axis in tumor progression was still not
elucidated. Human papillomavirus 16 (HPV16) was causally
linked to the carcinogenesis and progression in most of
the cervical cancers. It’s reported that the oncogene E6
from HPV16 could stimulate NF-κB activity in multiple
mechanisms. We demonstrated that E6 transfection
largely increased the transcription activity of NF-κB in
CaSki cells (Fig. 5a). Consistently, E6 transfection also
potentiated the binding activity between the p65 protein
and LPTS promoter in EMSA tests (Fig. 5b).
Subsequently, E6 transfection dramatically suppressed LPTS
expression in the mRNA and protein levels in a p65
dependent manner (Fig. 5c-d). We next explored the
functional relevance of E6-NF-κB axis by measuring
cancer cell growth in vitro and in xenograft models. E6
transfection significantly induced CaSki cell growth, and
this effect was abrogated by additional treatment of a
NF-κB inhibitor (Fig. 5e). Further, in subcutaneous
xenograft models, we confirmed that E6 transfection
potentiated NF-κB dependent CaSki tumor growth (Fig. 5f ).
NF-κB promotes LPTS-dependent growth of CaSki cells
To validate whether NF-κB regulates cervical cancer cell
growth through repressing LPTS, the p65-silenced CaSki
cells were additionally transfected with LPTS shRNAs.
In CCK8 assays, repressed cell growth by p65 silence
was largely rescued by LPTS knockdown (Fig. 6a).
Consistently, silence of p65 significantly inhibited the growth
of CaSki tumor, and this effect was prevented by
additional knockdown of LPTS by shRNAs (Fig. 6b). We
next investigated the survival time of the CaSki
tumorbearing mice, and demonstrated that p65 deficiency
notably improved the survival of those tumor-bearing mice
in a LPTS dependent manner (Fig. 6c).
NF-κB promotes LPTS-dependent growth of colorectal
Given the importance of NF-κB pathway in
], we presumed that NF-κB-LPTS axis might
play a dominant role not only in cervical cancer but also
in other inflammation-associated tumors, such as gastric
cancer and colon cancer. From the open database of
gene expression in human cancer tissues
(http://merav.wi.mit.edu/SearchByGenes.html), we found the
expression of p65 was increased, while the level of LPTS was
Fig. 6 NF-κB promotes LPTS-dependent growth of CaSki cells. a CCK8 assays of the CaSki cells. The PLKO or P65 shRNA-2-transfected CaSki cells
were additionally transfected with a human LPTS shRNA (hLPTS-KD). CCK8 assays were performed dynamically. (n = 5, **P < 0.01, means ± s.e.ms.,
Student’s t-test). b The size of the inoculated CaSki tumors. The CaSki cells were treated as described in (a), and injected subcutaneously in the
Balb/C nude mice. The tumor size was measured dynamically as indicated. (n = 10, *P < 0.05, means ± s.e.ms., Student’s t-test). c Percent survival
of the CaSki tumor-bearing mice. The Balb/C nude mice were inoculated with subcutaneous CaSki tumors as described in (b), and the survival
time was recorded. (n = 10, **P < 0.01, means ± s.e.ms., Gehan-Breslow-Wilcoxon test)
reduced in colon cancer tissues relative to the normal
colons (Fig. 7a). A similar expression profile of p65 and
LPTS as what we found in the colon cancer was also
observed in gastric cancer tissues and normal ones (Fig. 7b).
To further confirm the role of NF-κB-LPTS axis in cancer
cell growth, we selected a mouse colorectal cancer cell line
MC-38 and set up a subcutaneous xenograft model on
C57BL/6 mice. In vitro, we verified that p65 silence
potentiated LPTS expression in MC-38 cells (Fig. 7c-d).
LPTS knockdown could fully rescue p65
silenceattenuated cell growth (Fig. 7e). In vivo, we demonstrated
that p65 deficiency notably inhibited MC-38 tumor
growth and improved the survival of those tumor-bearing
mice in a LPTS dependent way (Fig. 7f-g). Furthermore, a
human gastric cancer cell line MKN-45 was employed to
confirm the function of NF-κB-LPTS axis in cancer cell
growth. As expected, similar results as what we found in
MC-38 cells were obtained in CCK8 assays and
subcutaneous xenograft models (Fig. 7h-j).
Chronic inflammation and LPTS expression are
associated with cancer development [
]. However, the
relationship between inflammation and LPTS expression is
still not known. In the present study, we for the first
time demonstrated that NF-κB (p65) inhibited LPTS
expression by directly binding to two consensus response
elements in the promoter region of LPTS. We also
revealed that NF-κB (p65) potentiated LPTS-dependent
cancer cell growth in vitro and in xenograt models.
Clinical relevance study indicated that NF-κB-LPTS axis
might be a potential target in cancer treatment.
NF-κB and JNK signaling are classic proinflammatory
pathways in response to inflammatory stimuli [
In the present study, we investigated those two pathways
and identified NF-κB (p65) as a functional suppressor of
LPTS expression. In fact, other inflammatory pathways
(e.g. STAT signals  and inflammasomes [
needed to be investigated in future studies. Perhaps, we
could identify more inflammation response elements in
the LPTS promoter.
Whether LPTS would regulate inflammation signals is
another interesting question. According to our data
shown in Fig. 7c, LPTS silence didn’t affect NF-κB p65
expression notably. Meanwhile, the transcription activity
of NF-κB was also not regulated by LPTS (data not
shown). Those findings indicated that LPTS didn’t
regulate NF-κB signaling. However, whether and how the
inflammatory cytokines (e.g. IL-1β, IL-6, TNFα and IL-10)
or other inflammatory signals (e.g. JNK, Stat and
inflammasome pathways) are influenced by LTPS still need to
be investigated in future studies. High throughput
studies like gene expression arrays or RNA sequencing
would be helpful in answering the above questions.
LPTS, a potent inhibitor of telomerase activity, is a
well-characterized tumor suppressor [
inhibits proliferation, migration and invasion of cancer
cells . Reduced expression of LPTS correlates to the
progressive features of cancers [
]. To the best of our
knowledge, no drugs have been designed to modulate
the expression of LPTS. Therefore, identifying the
regulators of LPTS is essential for exploring novel targets for
cancer therap. Here we identified that LPTS was
negatively regulated by NF-κB, which could be inhibited by a
series of specific inhibitors (e.g. BAY11–7082). Our
findings indicated that the NF-κB inhibitors might be
translated in cancer therapy. Indeed, emerging studies
reported that modulation of LPTS expression could
synergize the anti-tumor effects of regular chemotherapy
drugs in clinic [
It should be pointed out that not all the LPTS
deficiency in cancers was resulted from inflammation
stimuli. As shown in Fig. 1, LPTS expression was increased
in breast and prostate cancer tissues. These findings
revealed a complex regulatory mechanism of LPTS
expression. As reported previously in cervical cancer cells, we
demonstrated that LPTS was also a target gene of p53
], a transcription factor which was generally
inactivated in multiple cancer cells [
]. It’s very important
to identify which factor (e.g. p65, p53 and other
regulators) is more dominant in a specific type of cancer. In
fact, p53 was usually inactive due to gene mutation ,
while p65 was activated in response to the inflammatory
tumor microenvironment [
]. Both the genetic and
environment factors would regulate LPTS-dependent
Taken together, we identified LPTS as a novel target of
the transcription factor NF-κB. Modulation of
NF-κBLPTS axis might represent a promising target in cancer
CCK8: Cell counting kit-8; ChIP: Chromatin immunoprecipitation;
EMSA: Electrophoretic mobility shift assay; FBS: Fetal bovine serum;
HPV16: Human papillomavirus type 16; hTERT: human telomerase reverse
transcriptase; IL-10: Interleukin-10; IL-1β: Interleukin-1β; IL-6: Interleukin-6;
JNK: c-Jun NH2-terminal protein kinase; NF-κB: Nuclear factor-kappaB;
PINX1: PIN2/TERF1-interacting Telomerase Inhibitor 1; qPCR: quantitative
polymerase chain reaction; TNFα: Tumor necrosis factor α
This work was supported by grants (81,402,268 to D.L., and 31,401,103 to Y.Z.)
from the National Natural Science Foundation of China and by Foundation and
Frontier Research Project from Chongqing (cstc2017jcyjBX0071 to H.M).
Availability of data and materials
The data regarding to the expression of p65 and LPTS in cancer and normal
tissues were from an open database (http://merav.wi.mit.edu/SearchByGenes.html).
DL, HM and YZ conducted the experiments and analyzed the data. XK, SS,
WX, RS, AH, RW, KZ, YL, YM and HL contributed to discussion. HM and FH
were major guarantors of the present work and had full access to all the
data. HM integrated the data, wrote the manuscript and performed the
submission. All authors read and approved the final manuscript.
All the mouse experiments were approved by the Institutional Animal Care
and Use Committee of the Third Military Medical University and carried out
in accordance with the "Guide for the care and use of laboratory animals"
published by the US National Institutes of Health (Publication no.85–23,
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.
1. Fernandes JV , DEMF TA , DEA JC , Cobucci RN , DEC MG , Andrade VS , DEA JM. Link between chronic inflammation and human papillomavirus-induced carcinogenesis (review) . Oncol Lett . 2015 ; 9 : 1015 - 26 .
2. Gomes M , Teixeira AL , Coelho A , Araujo A , Medeiros R . The role of inflammation in lung cancer . Adv Exp Med Biol . 2014 ; 816 : 1 - 23 .
3. Izano M , Wei EK , Tai C , Swede H , Gregorich S , Harris TB , Klepin H , Satterfield S , Murphy R , Newman AB , et al. Chronic inflammation and risk of colorectal and other obesity-related cancers: the health, aging and body composition study . Int J Cancer . 2016 ; 138 : 1118 - 28 .
4. McKay CJ , Glen P , McMillan DC . Chronic inflammation and pancreatic cancer . Best Pract Res Clin Gastroenterol . 2008 ; 22 : 65 - 73 .
5. Qadri Q , Rasool R , Gulzar GM , Naqash S , Shah ZA. H. Pylori infection, inflammation and gastric cancer . J Gastrointest Cancer . 2014 ; 45 : 126 - 32 .
6. Yang H , Qi H , Ren J , Cui J , Li Z , Waldum HL , Cui G . Involvement of NFkappaB/IL-6 pathway in the processing of colorectal carcinogenesis in colitis mice . Int J Inflam . 2014 ; 2014 : 130981 .
7. Merga YJ , O'Hara A , Burkitt MD , Duckworth CA , Probert CS , Campbell BJ , Pritchard DM . Importance of the alternative NF-kappaB activation pathway in inflammation-associated gastrointestinal carcinogenesis . Am J Physiol Gastrointest Liver Physiol . 2016 ; 310 : G1081 - 90 .
8. Wang J , Ni WH , KB H , Zhai XY , Xie F , Jie J , Zhang NN , Jiang LN , Yuan HY , Tai GX . Targeting MUC1 and JNK by RNA interference and inhibitor inhibit the development of hepatocellular carcinoma . Cancer Sci . 2017 ; 108 : 504 - 11 .
9. Kulkarni SS , Vastrad PP , Kulkarni BB , Markande AR , Kadakol GS , Hiremath SV , Kaliwal S , Patil BR , Gai PB . Prevalence and distribution of high risk human papillomavirus (HPV) types 16 and 18 in carcinoma of cervix, saliva of patients with oral squamous cell carcinoma and in the general population in Karnataka, India . Asian Pac J Cancer Prev . 2011 ; 12 : 645 - 8 .
10. Ohata H , Kitauchi S , Yoshimura N , Mugitani K , Iwane M , Nakamura H , Yoshikawa A , Yanaoka K , Arii K , Tamai H , et al. Progression of chronic atrophic gastritis associated with helicobacter pylori infection increases risk of gastric cancer . Int J Cancer . 2004 ; 109 : 138 - 43 .
11. Flemer B , Lynch DB , Brown JM , Jeffery IB , Ryan FJ , Claesson MJ , O'Riordain M , Shanahan F , O'Toole PW . Tumour-associated and non-tumour-associated microbiota in colorectal cancer . Gut . 2017 ; 66 : 633 - 43 .
12. Danese S , Mantovani A . Inflammatory bowel disease and intestinal cancer: a paradigm of the yin-Yang interplay between inflammation and cancer . Oncogene . 2010 ; 29 : 3313 - 23 .
13. Umezawa K. Inhibition of tumor growth by NF-kappaB inhibitors . Cancer Sci . 2006 ; 97 : 990 - 5 .
14. Xia C , Watton S , Nagl S , Samuel J , Lovegrove J , Cheshire J , Woo P . Novel sites in the p65 subunit of NF-kappaB interact with TFIIB to facilitate NFkappaB induced transcription . FEBS Lett . 2004 ; 561 : 217 - 22 .
15. Tsuchiya Y , Asano T , Nakayama K , Kato T Jr, Karin M , Kamata H. Nuclear IKKbeta is an adaptor protein for IkappaBalpha ubiquitination and degradation in UV-induced NF-kappaB activation . Mol Cell . 2010 ; 39 : 570 - 82 .
16. Ganchi PA , Sun SC , Greene WC , Ballard DW. I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding . Mol Biol Cell . 1992 ; 3 : 1339 - 52 .
17. Zhou XZ , KP L. The Pin2/TRF1-interacting protein PinX1 is a potent telomerase inhibitor . Cell . 2001 ; 107 : 347 - 59 .
18. Soohoo CY , Shi R , Lee TH , Huang P , KP L , Zhou XZ . Telomerase inhibitor PinX1 provides a link between TRF1 and telomerase to prevent telomere elongation . J Biol Chem . 2011 ; 286 : 3894 - 906 .
19. Liao C , Zhao M , Song H , Uchida K , Yokoyama KK , Li T . Identification of the gene for a novel liver-related putative tumor suppressor at a high-frequency loss of heterozygosity region of chromosome 8p23 in human hepatocellular carcinoma . Hepatology . 2000 ; 32 : 721 - 7 .
20. Qian D , Cheng J , Ding X , Chen X , Guan Y , Zhang B , Wang J. Er P , Qiu M , Zeng X , et al: PinX1 suppresses tumorigenesis by negatively regulating telomerase/telomeres in colorectal carcinoma cells and is a promising molecular marker for patient prognosis . Onco Targets Ther . 2016 ; 9 : 4821 - 31 .
21. Shi R , Zhao Z , Zhou H , Wei M , Ma WL , Zhou JY , Tan WL . Reduced expression of PinX1 correlates to progressive features in patients with prostate cancer . Cancer Cell Int . 2014 ; 14 : 46 .
22. Banik SS , Counter CM . Characterization of interactions between PinX1 and human telomerase subunits hTERT and hTR . J Biol Chem . 2004 ; 279 : 51745 - 8 .
23. Wang HB , Wang XW , Zhou G , Wang WQ , Sun YG , Yang SM , Fang DC . PinX1 inhibits telomerase activity in gastric cancer cells through Mad1/c-Myc pathway . J Gastrointest Surg . 2010 ; 14 : 1227 - 34 .
24. Dai R , Iwama A , Wang S , Kapila YL . Disease-associated fibronectin matrix fragments trigger anoikis of human primary ligament cells: p53 and c-myc are suppressed . Apoptosis . 2005 ; 10 : 503 - 12 .
25. Wu G , Liu D , Jiang K , Zhang L , Zeng Y , Zhou P , Zhong D , Gao M , He F , Zheng Y. PinX1, a novel target gene of p53, is suppressed by HPV16 E6 in cervical cancer cells . Biochim Biophys Acta . 1839 ; 2014 : 88 - 96 .
26. An J , Mo D , Liu H , Veena MS , Srivatsan ES , Massoumi R , Rettig MB . Inactivation of the CYLD deubiquitinase by HPV E6 mediates hypoxia-induced NF-kappaB activation . Cancer Cell . 2008 ; 14 : 394 - 407 .
27. Miao H , Ou J , Peng Y , Zhang X , Chen Y , Hao L , Xie G , Wang Z , Pang X , Ruan Z , et al. Macrophage ABHD5 promotes colorectal cancer growth by suppressing spermidine production by SRM . Nat Commun . 2016 ; 7 : 11716 .
28. Miao H , Zhang Y , Lu Z , Yu L , Gan L . FOXO1 increases CCL20 to promote NFkappaB-dependent lymphocyte chemotaxis . Mol Endocrinol . 2012 ; 26 : 423 - 37 .
29. Livak KJ , Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method . Methods . 2001 ; 25 : 402 - 8 .
30. Bakiri L , Hamacher R , Grana O , Guio-Carrion A , Campos-Olivas R , Martinez L , Dienes HP , Thomsen MK , Hasenfuss SC , Wagner EF . Liver carcinogenesis by FOS-dependent inflammation and cholesterol dysregulation . J Exp Med . 2017 ; 214 : 1387 - 409 .
31. Brennan CA , Garrett WS . Gut microbiota, inflammation, and colorectal cancer . Annu Rev Microbiol . 2016 ; 70 : 395 - 411 .
32. Deivendran S , Marzook KH , Radhakrishna Pillai M. The role of inflammation in cervical cancer . Adv Exp Med Biol . 2014 ; 816 : 377 - 99 .
33. Wessler S , Krisch LM , Elmer DP , Aberger F . From inflammation to gastric cancer - the importance of hedgehog/GLI signaling in helicobacter pyloriinduced chronic inflammatory and neoplastic diseases . Cell Commun Signal . 2017 ; 15 : 15 .
34. Kim JK , Park GM . Indirubin-3 -monoxime exhibits anti-inflammatory properties by down-regulating NF-kappaB and JNK signaling pathways in lipopolysaccharide-treated RAW264.7 cells . Inflamm Res . 2012 ; 61 : 319 - 25 .
35. Textor S , Accardi R , Havlova T , Hussain I , Sylla BS , Gissmann L , Cerwenka A . NF-kappa B-dependent upregulation of ICAM-1 by HPV16-E6/E7 facilitates NK cell/target cell interaction . Int J Cancer . 2011 ; 128 : 1104 - 13 .
36. Egan LJ , Toruner M. NF-kappaB signaling: pros and cons of altering NFkappaB as a therapeutic approach . Ann N Y Acad Sci . 2006 ; 1072 : 114 - 22 .
37. Miao H , Ou J , Ma Y , Guo F , Yang Z , Wiggins M , Liu C , Song W , Han X , Wang M , et al. Macrophage CGI-58 deficiency activates ROS-inflammasome pathway to promote insulin resistance in mice . Cell Rep . 2014 ; 7 : 223 - 35 .
38. Horvath CM . STAT proteins and transcriptional responses to extracellular signals . Trends Biochem Sci . 2000 ; 25 : 496 - 502 .
39. Baichuan L , Cao S , Liu Y. LPTS : a novel tumor suppressor gene and a promising drug target for cancer intervention . Recent Pat Anticancer Drug Discov . 2015 ; 10 : 170 - 5 .
40. Mei PJ , Chen YS , Du Y , Bai J , Zheng JN . PinX1 inhibits cell proliferation, migration and invasion in glioma cells . Med Oncol . 2015 ; 32 : 73 .
41. Deng W , Jiao N , Li N , Wan X , Luo S , Zhang Y. Decreased expression of PinX1 protein predicts poor prognosis of colorectal cancer patients receiving 5-FU adjuvant chemotherapy . Biomed Pharmacother . 2015 ; 73 : 1 - 5 .
42. JH X , SL H , Shen GD , Shen G . Tumor suppressor genes and their underlying interactions in paclitaxel resistance in cancer therapy . Cancer Cell Int . 2016 ; 16 : 13 .
43. Sur S , Pagliarini R , Bunz F , Rago C , Diaz LA , Jr. , Kinzler KW , Vogelstein B , Papadopoulos N : A panel of isogenic human cancer cells suggests a therapeutic approach for cancers with inactivated p53 . Proc Natl Acad Sci U S A 2009 , 106 : 3964 - 3969 .
44. Meuwissen R , Linn SC , Linnoila RI , Zevenhoven J , Mooi WJ , Berns A . Induction of small cell lung cancer by somatic inactivation of both Trp53 and Rb1 in a conditional mouse model . Cancer Cell . 2003 ; 4 : 181 - 9 .
45. Smith ML , Fornace AJ , Jr.: Genomic instability and the role of p53 mutations in cancer cells . Curr Opin Oncol 1995 , 7 : 69 - 75 .
46. Schottelius AJ , Dinter H. Cytokines , NF-kappaB, microenvironment, intestinal inflammation and cancer . Cancer Treat Res . 2006 ; 130 : 67 - 87 .