Autocrine parathyroid hormone-like hormone promotes intrahepatic cholangiocarcinoma cell proliferation via increased ERK/JNK-ATF2-cyclinD1 signaling
Tang et al. J Transl Med
Autocrine parathyroid hormone-like hormone promotes intrahepatic cholangiocarcinoma cell proliferation via increased ERK/JNK-ATF2-cyclinD1 signaling
Jing Tang 0 1
Yan Liao 0 1
Shuying He 0 1
Jie Shi 0 1
Liang Peng 0 1
Xiaoping Xu 1
Fang Xie 1
Na Diao 1
Jinlan Huang 3
Qian Xie 1
Chuang Lin 4
Xiaoying Luo 1
Kaili Liao 1
Juanjuan Ma 2
Jingyi Li 1
Daichao Zhou 1
Zhijun Li 1
Jun Xu 1
Chao Zhong 1
Guozhen Wang 1
Lan Bai 1
0 Jing Tang , Yan Liao, Shuying He, Jie Shi and Liang Peng contributed
1 Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University , No. 1838, Guangzhou Avenue North, Baiyun District, Guangzhou, Guangdong , China
2 Department of Gastroenterology, Dali Bai Autonomous Prefecture People's Hospital , Dali, Yunnan , China
3 Laboratory Medicine Center, Nanfang Hospital, Southern Medical University , Guangzhou, Guangdong , China
4 Department of Pathology, Nanfang Hospital, Southern Medical University , Guangzhou, Guangdong , China
Background and aims: Intrahepatic cholangiocarcinoma (ICC) is an aggressive tumor with a high fatality rate. It was recently found that parathyroid hormone-like hormone (PTHLH) was frequently overexpressed in ICC compared with non-tumor tissue. This study aimed to elucidate the underlying mechanisms of PTHLH in ICC development. Methods: The CCK-8 assay, colony formation assays, flow cytometry and a xenograft model were used to examine the role of PTHLH in ICC cells proliferation. Immunohistochemistry (IHC) and western blot assays were used to detect target proteins. Luciferase reporter, chromatin immunoprecipitation (ChIP) and DNA pull-down assays were used to verify the transcription regulation of activating transcription factor-2 (ATF2). Results: PTHLH was significantly upregulated in ICC compared with adjacent and normal tissues. Upregulation of PTHLH indicated a poor pathological differentiation and intrahepatic metastasis. Functional study demonstrated that PTHLH silencing markedly suppressed ICC cells growth, while specific overexpression of PTHLH has the opposite effect. Mechanistically, secreted PTHLH could promote ICC cell growth by activating extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and subsequently upregulated ATF2 and cyclinD1 expression. Further study found that the promoter activity of PTHLH were negatively regulated by ATF2, indicating that a negative feedback loop exists. Conclusions: Our findings demonstrated that the ICC-secreted PTHLH plays a characteristic growth-promoting role through activating the canonical ERK/JNK-ATF2-cyclinD1 signaling pathways in ICC development. We identified a negative feedback loop formed by ATF2 and PTHLH. In this study, we explored the therapeutic implication for ICC patients.
Parathyroid hormone-like hormone; Activating transcription factor-2; Proliferation; Intrahepatic cholangiocarcinoma
Background
Intrahepatic cholangiocarcinoma arises from epithelial
cells lining the bile duct and occurs proximally at the
second degree bile ducts within the liver. The condition is
commonly associated with cirrhosis, viral hepatitis B and
C [1]. ICC displays a feature of rapid progression and a
poor outcome, and its global disease incidence has been
rapidly increasing [2]. Radical resection and curative liver
transplantation are preferred surgical treatments for ICC,
however, the patients with vascular and lymph nodes
metastases are not eligible for surgical therapy. Although
the chemotherapy regimen of gemcitabine and cisplatin
and locoregional therapies are additional options for
inoperable ICC patients, the 5-year survival rates are very
low. An enhanced understanding of the biology
pathological progress and the interaction with tumor
microenvironment of ICC is needed to improve patient survival.
PTHLH, also referred to parathyroid hormone-related
protein (PTHrP), has emerged as an important cytokine
with diverse cell functions, including growth, survival,
migration, and differentiation [3]. Tumor-derived PTHLH
participates in the bone metastatic processes of breast
cancer via an intracrine fashion [4]. In addition, PTHLH
supports colorectal cancer cell to form distant lung
metastatic processes via inducing caspase-independent death in
human lung vasculature endothelial cells [5]. A previous
report demonstrated that PTHLH produced by
proliferating bile duct epithelial cells and may interact with growth
factors and hormones to form complex loops that
promotes proliferation [6]. Growing evidences indicate that
PTHLH-producing cholangiocarcinoma (CHO) patients
suffer from humoral hypercalcemia of malignancy [7–9],
but litter is known regarding PTHLH’s effect on ICC cells
growth. MAP kinase pathways are involved in the
process through the PTHLH-induced activation of PTH1R to
activate downstream effectors [10–12]. ATF2, as a
downstream effector of MAPK in response to cytokines, is
phosphorylated on Thr69 and/or Thr71 by either JNK or p38
[13, 14], and is also activated by the ERK1/2 pathway in
two step [13]. Several observations support that ATF2
regulates cell cycle progression via controlling the
transcriptional output of several key genes, including CyclinD1,
CyclinA and RB1 [15–18]. However, no studies have
documented a role for the PTHLH-MAPK-ATF2-CyclinD1
signaling axis in the regulation of ICC cells growth. This
study aims to elucidate the role and clinical significance of
the PTHLH-MAPK-ATF2-CyclinD1 axis in ICC cell cycle
progression.
Methods
Patients, tissue samples and microarrays
59 ICC samples and paired non-tumor tissues and 10
normal tissues were obtained from the Department of
Hepatobiliary Surgery, NanFang Hospital, Southern
Medical University between 2014 and 2016. All patients
signed informed consent for therapy and subsequent
tissue studies, which were approved by the NanFang
Hospital Institutional Review Board. The ICC tissue
microarrays, which contained 100 cases, and the
extrahepatic cholangiocarcinoma (ECC) tissue microarrays,
which contained 27 cases were purchased from Shanghai
Outdo Biotech Inc. (Shanghai, China). All tumors were
defined as a primary tumor arising from the bile ducts
and diagnosed as adenocarcinomas. Tumor stage was
defined according to the seventh edition of American
Joint Committee on Cancer/International Union against
Cancer (AJCC/UICC). All specimens were used for
routine pathological processing with comparable
clinicopathological features, and complete follow-up data were
obtained.
Western blot, real‑time PCR analysis, immunohistochemistry and immunofluorescence
RNA and protein lysate extraction, cDNA synthesis, final
real-time PCR and western blots were performed
according to general protocols. ICC cells were processed for
immunofluorescence (IF) using target antibodies with
optimized conditions. In addition, human samples and
ICC microarrays were subjected to IHC staining to
evaluate the expression of relative proteins.
Cell counting kit‑8 assay, colony formation assays, cell cycle analysis, cell migration and invasion assay
Cell counting kit-8 assay, colony formation assays, cell
cycle analysis, cell migration and invasion assay and
Annexin V apoptosis assay were performed according to
general protocols and can be found in Additional file 1.
Dual‑luciferase reporter gene assay
To determine the effect of ATF2 on PTHLH
transcription, RBE cells were transfected with pGL3 as vehicle
control, pGL3-PTHLH or pGL3-MUT-PTHLH using
Lipofectamine 3000. Firefly and Renilla luciferase
activities were measured separately on a fluorescence
spectrophotometer (FlOUstar omega, BMG Labtech, Germany)
in triplicate according to the manufacturer’s instructions
for the dual-luciferase reporter assay kit (Promega). The
relative transcriptional activity was normalized by the
corresponding vehicle control value.
Chromatin immunoprecipitation (ChIP) assay
Genomic DNA prepared from RBE cells transfected with
shControl was crosslinked with 1% formaldehyde and
fragmented into 500 ± 100-bp fragments by sonication.
Soluble chromatin was then incubated overnight with
anti-ATF2 antibodies. Finally, the immunoprecipitated
DNA fragments were amplified and quantified using
realtime PCR using the following PCR primers specific to the
human PTHLH promoter region.
Establishment of a subcutaneous tumor xenograft
RBE cells (shCtrl or shPTHLHx) (1 × 107) were injected
subcutaneously into the groins of BALB/c nude mice
(6 weeks old, male, n = 5 for each group). Tumor growth
was monitored at 2 or 3-day intervals. When the mice
were sacrificed after 25 days, tumor weight and size were
measured, and the tumor was fixed for additional
experimental use.
Statistical analyses
Different statistical analysis methods were used to
compare different groups or different categories of data.
Extended details regarding materials and methods can be
found in Additional file 1.
Results
PTHLH is highly expressed in human CHO tissues specimens and ICC cell lines
To identify the potential role of PTHLH in CHO, we
evaluated 59 ICC samples and paired non-tumor tissues
from NanFang Hospital. We also screened an additional
10 samples of normal liver tissues for comparison. IHC
analyses of ICC tumor regions revealed strong staining
of PTHLH compared with that in adjacent regions in the
same patients (Fig. 1a, top panel and b).
Immunostaining of PTHLP protein was located in the cytoplasm and
nucleus of ICC cells. We also observed weak staining
in the bile duct of adjacent and normal tissue samples
(Fig. 1a, top panel). In addition, we detected the
expression of PTH1R, a specific receptor for PTHLH, in
membranes of ICC cells (Fig. 1a, middle panel). Cytokeratin19
(CK) staining revealed the presence of adenocarcinoma
cells and biliary epithelial cells (Fig. 1a, bottom panel). In
addition, to further confirm the expression of PTHLH in
CHO, we screened ICC microarrays that contained 100
cases and the ECC microarrays that contained 27 cases
(Fig. 1c and Additional file 1: Figure S1). Our results
were consistent with the conclusion above that PTHLH
was highly expressed in CHO cells. In addition, PTHLH
protein expression was examined in ICC cell lines by
IF microscopy (Fig. 1e). Microscopy analysis detected
cytosolic and nucleus expression of PTHLH in RBE and
HCCC-9810 cells, which is consistent with previous
observations.
between clinicopathological features and PTHLH
expression levels in CHO cases. These patients were divided
into high- (score, 2–3) or low- (score, 0–1) PTHLH
expression groups according to the immunostaining
scores (Fig. 1c, d). Scoring was conducted according to
the ratio and intensity of positive-staining cells: 0–5%
scored 0; 6–35% scored 1; 36–70% scored 2; more than
70% scored 3. The final score was designated as low or
high expression group as follows: score 0–1, low
expression, score 2–3, high expression. A high expression of
PTHLH was positively correlated with poor pathological
differentiation (p < 0.05) (Table 1). These findings
indicate that PTHLH expression might contribute to ICC
progression and be a potential therapeutic target of this
disease.
PTHLH promotes ICC cells growth
The above data suggested that PTHLH may play a
critical role in ICC progression. To address whether PTHLH
affects cell proliferation, we first investigated endogenous
PTHLH levels in tow ICC cell lines. We observed they
both have PTHLH endogenous expression (Additional
file 1: Figure S2A). We then generated two
PTHLH-specific shRNAs to silence the endogenous PTHLH
expression in ICC cells. shPTHLHx, which induced the most
significant knock-down (KD) effect, was used for vivo
study. We stably depleted PTHLH in RBE and
HCCC9810 cells. The relative expression of PTHLH in RBE and
HCCC-9810 cells was confirmed by qPCR and western
blot (Additional file 1: Figure S2B, C). PTHLH
depletion significantly decreased ICC cell proliferation (Fig. 2a
and Additional file 1: Figure S3). To evaluate the effects
of PTHLH re-expression on tumor growth in vitro, we
knock-down endogenous PTHLH and then reintroduced
lentivirus-mediated vector (LV-Ctrl) and PTHLH using
lentivirus-mediated PTHLHGFP (LV-PTHLHRE) to
examine whether the re-expression of PTHLH could rescue
the retarded proliferation (Additional file 1: Figure S2D).
Compared with the control, incubation with
PTHLHspecific shRNA resulted in elongated morphology cells
and less confluent cell growth. When exposed to
lentivirus-mediated PTHLHGFP, cell growth returned to normal
(Additional file 1: Figure S3). Furthermore, we observed
that PTHLH secretion was upregulated upon the
reintroduction of PTHLH, indicating an autocrine function of
PTHLH (Additional file 1: Figure S2E). Similarly, PTHLH
re-expression increased LV-PTHLHRE ICC cell
proliferation (Fig. 2b and Additional file 1: Figure S3).
Overexpression PTHLH is positively correlated with poor pathological differentiation in ICC patients
To investigate the clinical significance of PTHLH
upregulation in CHO, we further analyzed the relationship
PTHLH alters the cell cycle distribution
Cell cycle distribution was analyzed by flow cytometry
analysis to determine whether PTHLH enhances cell
growth and promotes tumorigenesis via an alteration of
the cell cycle. After 12 h of serum starvation for
synchronization, the cell population in the G2/M phase is
significantly increased upon PTHLH re-expression, whereas
the G0/G1 and S phase cell population remained more
constant in RBE cells (Fig. 2c top panel). In contrast, the
reverse effect was observed when PTHLH was depleted.
shPTHLH arrested RBE cells at the G0/G1 phase, and the
proportion of cells in the S and G2/M phase decreased
(Fig. 2c bottom panel). These results demonstrate that
PTHLH re-expression facilitates the S to G2/M phase
transition. However, PTHLH deletion blocks the cell
cycle by inhibiting the G0/G1 to S phase transition. The
similar results were obtained from another ICC cell line,
HCCC-9810 (Fig. 2d). To further explore the molecular
basis of PTHLH-enhanced tumour development, we
investigated the roles of PTHLH on metastasis using
in vitro migration and Matrigel invasion assays. The
results indicate that PTHLH facilitating RBE cells
migration not invasion (Additional file 1: Figure S4A). We also
found no significant differences between migration and
invasion of HCCC-9810 cells (Additional file 1: Figure
S4B). We quantitatively investigated the effect of PTHLH
on apoptosis by flow cytometry after staining with
Annexin V and 7-amino-actinomycin. The results
indicate that ICC cell apoptosis is not regulated by PTHLH
(Additional file 1: Figure S4C).
PTHLH regulates the expression of genes controlling the cell cycle
The observed differences in the cell cycle distribution
were due to the different expression levels of key cell
cycle proteins. We noted an increased accumulation of
G2/M-phase cells upon PTHLH re-expression compared
with that in LV-Ctrl cells. We observed that p-cdc2
protein levels decreased significantly, whereas CyclinB1
levels remains constant when PTHLH was re-expressed
(Fig. 2e, left panel). These results indicate that PTHLH
re-expression promotes RBE cells mitosis via
downregulating p-cdc2 expression. We also detected G0/G1
phaserelated proteins (CDK4/CyclinD1 and CDK6/Cyclin
D3). CDK4/CyclinD1 protein levels increased slightly,
whereas CDK6 and Cyclin D3 levels remained constant
(Fig. 2e, left panel). Given that the cell cycle was altered
by PTHLH depletion, we focused our attention on key
proteins (CDK4/CyclinD1 and CDK6/Cyclin D3)
during the G0/G1 phase. Western blot analysis indicated that
CyclinD1 and CDK4 protein levels decreased
dramatically when PTHLH expression was deleted in RBE cells
(Fig. 2e, right panel). In contrast, Cyclin D3 and CDK6
expression remained constant. These data suggested that
PTHLH regulates the expression of cell cycle-related
proteins.
Loss of PTHLH expression suppresses tumorigenesis in vivo
To investigate whether PTHLH deletion suppresses
tumorigenesis in vivo, PTHLH-KD RBE cells (shPTHLHx)
were implanted subcutaneously into the right inguen,
and vector cells (shCtrl) were implanted into the left
inguen of nude mice (n = 5) (Fig. 3a). Tumor growth
was monitored as described in Additional file 1.
Consistent with the cell proliferation assay in vitro, tumor
growth was significantly decreased in mouse xenografts
with shPTHLHx compared with that of shCtrl (Fig. 3b,
c). Consistently, the nuclear expression of Ki-67, Cyclin
D1 and CDK4 proteins was significantly increased in the
shCtrl-RBE tumors compared with that in the
shPTHLHx-RBE tumors (Fig. 3e), which is consistent with the
in vitro study using western blot (Fig. 2e). These results
collectively suggest that PTHLH promotes ICC cell
proliferation.
PTHLH altered cell cycle genes via activating phosphorylated ATF2 through JNK/ERK1/2 signaling pathways
Accumulating studies highlight PTHLH as a cellular
cytokine with actions involved in both cell growth and
differentiation [3]. Previous reports supported that the
PTHLH can trigger the MAPK signaling cascade by
binding with PTH1R, which interacts with the MAPK
scaffolding protein β-arrestin2 and G-protein [10–12].
As a downstream effector of MAPK, ATF2 regulates cell
cycle progression through the transcriptional control of
CyclinD1 (Fig. 4a) [18]. We hypothesized that PTHLH
can increase ATF2 transcriptional activity by
activating ERK1/2 and JNK signaling cascades. As shown in
(Fig. 4b, c), we treated RBE cells with PTHLH (
1–34
)
recombination fragment and assessed the ATF2
expression. PTHLH (
1–34
) induced a time- and
dose-responsive increase of in ATF2 protein expression. In contrast,
the effect of PTHLH was attenuated at 100 nM (Fig. 4b,
dotted line). These results indicated that PTHLH exhibits
dose-dependent biphasic effects on ICC cell dynamics.
We also found endogenous PTHLH re-expression
upregulated ATF2 protein expression (Additional file 1: Figure
S5). In response to PTHLH stimulation, PTHLH/PTH1R
signaling triggers JNK and ERK1/2 signaling pathways
(Fig. 4d). Therefore, pharmacological approaches were
used to confirm that ATF2 transcriptional activity is
regulated by PTHLH. When RBE cells were pretreated
with an MEK1/2 inhibitor (U0126) or JNK1/2 inhibitor
(SP600125) for 1 h followed by PTHLH (
1–34
) treatment
for 4 h, we observed that the MEK1/2 and JNK1/2
inhibitor abrogated the PTHLH-induced phosphorylation of
ATF2 (p-ATF2) (Fig. 4e), suggesting that p-ATF2
induction involves the PTHLH-JNK/ERK1/2 signaling cascade.
Interestingly, we further found that the MEK inhibitor
U0126 and the JNK inhibitor SP600125 inhibited RBE
cell growth, arresting cells at the G0/G1 phase (Fig. 4e).
These results suggesting that the inhibition of JNK/
ERK1/2 attenuated PTHLH-induced ICC growth.
ATF2 negatively regulate PTHLH expression
ATF2 is a bZIP transcription factors which has an
ability to bind to the CRE consensus. According to previous
reports, the pthlh gene contains a CRE element within its
promoter region. Interestingly, we also found that ATF2
might interact with PTHLH promoter elements in
bioinformatics prediction methods (Additional file 1: Figure
S5). Next, we mapped the ATF2 response element(s) on
the PTHLH promoter. Analysis of the proximal region
revealed the presence of ATF2 target sequences at
positions − 2210 to − 2243 (Site #3) (Fig. 5a). Further
support for the role of ATF2 in the regulation of PTHLH
transcription was provided by ChIP analysis. Sheared
chromatin was immunoprecipitated with antibodies
to ATF-2 (or control IgG) followed by the PCR
amplification of PTHLH promoter sequences.
Immunoprecipitation of ATF2 enabled the amplification of PTHLH
promoter sequences, demonstrating the in vivo binding
of ATF2 to the PTHLH promoter. DNA pull-down assays
demonstrated ATF2 binding to the PTHLH promoter
region (Fig. 5b). Consistent with this finding, a
mutation within this site attenuated the basal level of reporter
activity and the binding of ATF2 (Fig. 5c), confirming
that ATF2 regulates PTHLH transcription via
binding its response element at the Site #3. Our studies also
indicated that PTHLH expression was regulated by
siATF2, as confirmed by qPCR and western blot (Fig. 5d).
To further confirm the correlation between PTHLH
and ATF2 expression in ICC, we detected ATF2
expression using the same samples (Fig. 5e). Further statistical
analysis revealed that the ATF2 expression correlated
with PTHLH expression in the tissue samples (r = 0.624,
p < 0.05), suggesting a potentially complicated regulatory
mechanism between PTHLH and ATF2.
Discussion
ICC is one of the most lethal epithelial cancers united
by poor diagnoses and adverse outcomes. The
condition frequently arises in the presence of chronic injury
and inflammation. Previous literatures documenting that
ICC is commonly associated with cirrhosis, viral
hepatitis B and C, and metabolic abnormalities [19–22]. The
molecular pathogenesis of ICC proliferation and
metastasis, as the main cause of ICC-related mortality, but the
mechanisms remain obscure. Our findings demonstrate
that PTHLH knockdown in ICC cells suppressed tumor
growth, while re-expression of PTHLH has the opposite
effect, highlighting the role of PTHLH as a critical
oncoprotein in ICC progression.
PTHLH/PTH1R signaling is aberrantly induced or
activated in different cancer types and is associated with
poor prognosis [5, 23–25]. In the present study, we found
that ICC cells produced PTHLH ligands that respond via
the expression of cognate receptors PTH1R, resulting in
the continuous activation of downstream signaling
pathways. Extensive evidence suggest that PTHLH is viewed
as a cellular cytokine, particularly in epithelial cancer
cells, that exhibits an autocrine or paracrine role in both
cell growth and differentiation [26, 27]. In our
previous study, we found that overexpression of the PTHLH
(LV-PTHLH), which transfects lentivirus-mediated
PTHLHGFP without deleting endogenous PTHLH
expression in ICC cells, may enter into the non-proliferative
cells (data not show). In contrast, LV-PTHLHRE ICC cells
promoted cell growth. Moreover, endogenous PTHLH
sustains the activation of MAPK signaling pathways, and
this effect was more pronounced after the addition of a
PTHLH recombination peptide. In contrast, the effect of
PTHLH on activation was attenuated at a higher
concentration (100 nM) compared with 50 nM (Fig. 5b, dotted
portion). These results clearly indicated that PTHLH may
function as a tumor cell growth promoter within a
certain concentration range. When the range is exceeding,
PTHLH becomes saturated and suppresses cells
proliferation. In our study, we also found re-expression PTHLH
promotes RBE cells migration and specific
overexpression PTHLH associates with intrahepatic metastasis in
ICC patients. All results indicated a potential ability of
facilitating tumor invasiveness. The data would suggest
that PTHLH may potentially transform ICC cells into
an aggressive form of the disease. And this also indicates
that PTHLH influences ICC cells growth and
differentiation with a low signal expression.
To investigate the effect of ICC secreted PTHLH on
cancer cell growth, we established an in-vitro PTHLH
secretion system. In our present work, we found that
PTHLH regulated cell growth by altering the cell cycle.
Cell cycle dysregulation is a major feature of
tumorigenesis, which occurs by shortening the G1 phase or
activating CDKs may favor tumor development [28]. Herein,
we demonstrate that PTHLH controlled cell cycle
progression. Secreted PTHLH protein act on target cells by
binding to its specific cell surface receptor: PTH1R. The
potential molecular mechanisms could be explained by
the finding that the PTHLH protein activates the JNK/
ERK1/2-ATF2 axis via interacting with the MAPK
scaffolding protein β-arrestin2, or triggering an early G
protein-dependent pathway meditated by PKA and PKC
[10] leading to cell cycle proliferation. Furthermore, we
provided evidence suggesting that PTHLH upregulates
ATF2 phosphorylation via activating the ERK1/2 and
JNK signaling pathways, which transcriptionally
upregulate CyclinD1 expression. When ERK1/2 and JNK are
pharmacologically inhibited in ICC cells (i.e., via U0126
and SP600125), which blocked PTHLH-induced
activation of ERK1/2 and JNK signal pathways, and
transcriptional activity of ATF2 (Fig. 6). Cyclin D1 is frequently
deregulated in cancer and is a biomarker of cancer
phenotypes and disease progression [29]. Previous findings
suggested that the deregulation of CyclinD1 expression
and CDK4 activation directly lead to some cancer
hallmarks by inducing proliferation [30–32]. We observed
that reduced CyclinD1 expression and CDK4 inactivation
directly inhibited proliferation, which is consistent with
previous reports. Consistently, our present work
demonstrated that PTHLH re-expression accelerated the G2
to M phase transition, which is similar to the effects of
numerous other oncogenes. Among the genes
functioning during the G2 and M phase transition, we observed
that the p-cdc2 levels were rapidly reduced upon PTHLH
re-expression. Several experimental findings indicate that
cdc2 is one of the master regulators of mitosis that
controls the centrosome cycle in complex with A- or B-type
cyclins [33]. Previous reports have suggested that cdc2
activity upon mitosis entry depends on p-cdc2 levels [34].
However, the reduction of cdc2 activity primarily drives
the exit from mitosis [33]. Our present study indicates
that PTHLH promote mitosis in ICC cells via
downregulating p-cdc2 expression. Interestingly, we observed
a paradoxical phenomenon that PTHLH overexpression
without knocking-down endogenous secretion arrests
ICC cells in the G1 phase and decreases CyclinD1
expression (data not show). We hypothesized that ostensibly
paradoxical responses between PTHLH deletion and
overexpression in cultured ICC cells appear to facilitate
a compromise between maximal mitogenic stimulation
and the avoidance of antiproliferative defenses.
Another interesting finding of this study is the negative
regulated role of ATF2 in RBE cells (Fig. 6). ATF2 is an
important transcription factor that can facilitate
malignant proliferation. In our previous study, we found that
ATF2 promoted growth of ICC cells and was correlated
with a poor prognosis for ICC patients. In vitro evidence
indicated that the upregulation of ATF2 phosphorylation
and activity promotes cancer progression via facilitating
cell proliferation-related gene expression. In our study, we
demonstrated that PTHLH can promote oncogenic
functions of ATF2 by activating ERK/JNK pathways. And a
previous report revealed PTHLH can activate PKC
pathway [35]. Eric Lau [36] previously reported that PKCε
promotes oncogenic functions of ATF2 in the nucleus while
blocking its apoptotic function at mitochondria. Thus,
we believe that PTHLH promotes nuclear
translocation and transcriptional function in the oncogenic
functions of ATF2. Interestingly, another novel finding of our
study is the negative role of ATF2 in PTHLH production
in REB cells. And we also found autocrine activation of
the PTHLH promoter by c-Jun (data not show).
Collectively, these findings indicated that ATF2 limits PTHLH
transcriptional output to maintain specific concentration
by forming a homodimer or a heterodimer with JUN. It
is possible that cancer cells may have negative feedback
loops that are essential for survival.
Conclusions
In summary, we report that ICC-secreted PTHLH
acts in an autocrine manner in intrahepatic
cholangiocarcinoma progression by activating the canonical
ERK/JNK signaling pathway. However, our findings
focus on only PTHLH-mediated ICC cell
proliferation, not provide new insight into the ICC metastasis.
Despite the importance of PTHLH tumorigenic role,
our knowledge of the PTH1R that mediate changes in
the tumor progression and interaction with PTHLH
in ICC is still limited. Based on our findings,
further investigation for interfering with PTH1R, which
mediate signaling in cancer cells, may serve as
effective treatment approaches to ICC patients. And we
will improve the mechanisms of
PTHLH/PTH1Rmediated ICC progression and involve the interaction
of the transcription factors CREB and AP-1 (c-JUN,
c-FOS and ATF2) in ICC development.
Additional file
Additional file 1. Additional Figures.
Abbreviations
ICC: intrahepatic cholangiocarcinoma; PTHLH: parathyroid hormone-like
hormone; PTHrP: parathyroid hormone-related protein; IHC:
immunohistochemistry; ChIP: chromatin immunoprecipitation; ATF2: activating transcription
factor-2; PTH1R: PTH type 1 receptor; ECC: extrahepatic cholangiocarcinoma;
IF: immunofluorescence; PI: propidium iodide; CK19: cytokeratin19; CHO:
cholangiocarcinoma; KD: knockdown.
Authors’ contributions
The first five authors contribute equally to this paper. JT assisted in the design
of study, performed experiments, analyzed/interpreted data, and drafted the
manuscript; YL, SH and LP contributed to study design, interpreted data, and
helped with manuscript revision; JS helped draft the manuscript; XX, FX, JH, XL
and JX provided technical support and helped to revise the manuscript; ZL,
CL, ND, KL, JM, GW, JL, DZ, CZ and QX helped performed some of the
experiments and supported materials; LB contributed to study design, revision of
the manuscript and provided funding. All authors read and approved the final
manuscript.
Acknowledgements
We thank AJE team for providing language help and writing assistance. This
work was supported by the National Natural Science Foundation of China
(Grants 81170354, 81470790, and 81500398), Natural Science Foundation of
Guangdong Province (2015A030313295 and 2015A030310480), Guangzhou
Pilot Project of Clinical and Translational Research Center (early gastrointestinal
cancers, No. 7415696196402), and Guangdong Provincial Bioengineering
Research Center for Gastroenterology Diseases.
Competing interests
The authors who have taken part in this study declared that they do not have
anything to disclose regarding funding or conflict of interest with respect to
this manuscript.
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated or
analyzed during the current study. Please contact author for data requests.
Consent for publication
The participants gave informed consent before taking part in this study. All
samples were de-identified.
Ethics approval and consent to participate
NanFang Hospital Institutional Review Board.
Institutional Animal Care Use Committee.
Funding
This work was supported by the National Natural Science Foundation of China
(Grants 81170354, 81470790, and 81500398), Natural Science Foundation of
Guangdong Province (2015A030313295 and 2015A030310480), Guangzhou
Pilot Project of Clinical and Translational Research Center (early gastrointestinal
cancers, No. 7415696196402), and Guangdong Provincial Bioengineering
Research Center for Gastroenterology Diseases.
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