Results
The
expression of thrombin in lung cancer is closely related
toclinicopathological
features and the prognosis of patients.
Thrombin has also been shown to contribute to tumor progression.
However,
the
expression
of thrombin in NSCLC tissues and the relationship between thrombin
expression and clinicopathological features and the prognosis of NSCLC
patients have not been reported. To confirm the presence of thrombin
(prothrombin) in
NSCLC,
132 patients with a pathologically confirmed diagnosis of NSCLC were
analyzed. We found the expression of thrombin was significantly
increased in tumors of all different types of NSCLC tissues compared
with their adjacent non-tumor lung tissues (Fig. 1A-C). There was no
significant difference in different subtypes of NSCLC (Fig. 1C). To
further evaluate the prognostic value of thrombin for NSCLC patients,
univariate and multivariate analyses were performed with the
clinicopathological characteristic. As shown in Table 1, thrombin
expression in tumor tissue was significantly correlated with TNM stage
of NSCLC.
The
5-year overall
survival
(OS) rates of thrombin-positive patients were significantly lower than
those of thrombin-negative patients (Fig. 1D). Moreover, both the mRNA
and protein levels were significantly increased in comparison to normal
lung cell line
(BEAS-2B)
detected by Q-PCR (Fig. 1E) and western blot (Fig. 1F) in three NSCLC
cell lines.
Thrombin
plays an important role in the progression of lung cancer.
To observe the role of thrombin in lung cancer cells, we constructed
A549THR-/- and
LLCThr-/- cells. Transwell assay results showed
that thrombin
depletion
inhibited cell migration in A549 and Lewis cells (Fig. 2A-D). IL6 has
been demonstrated to be involved in the development, progression and
metastasis in several cancers (Huang et al., 2018). Matrix
metalloproteinases (MMPs) affects the physical barrier of the tumor
microenvironment (TME) and induces metastasis (Lyu, Xiao, Yin, Yang &
He, 2019). We found that the expression levels of MMP9 and IL6 were
decreased in thrombin deficient cells, and the expression of MMP9 and
IL6 could be restored by adding exogenous thrombin (supplementary Fig.
S1).
To assess the role of thrombin in vivo, we employed tumor models. In
subcutaneous transplanted tumor model, tumor growth inhibition was
observed in
thrombin
deficient group
(Fig.
2E-G). In addition, the lung metastases were reduced in thrombin
deficient group (Fig. 2H-I). In orthotopic lung tumor model, thrombin
deficiency could markedly increase the survival time of mice (Fig. 2J).
And the control LLC cells generated massive lung tumor burden in mice.
Significantly, the depletion of thrombin led to smaller lung tumor
burden (Figure
2K).
HE staining of lung sections also revealed bigger tumor area in control
mice as compared to the thrombin deficient group (Figure 2L). Thrombin
deficiency also inhibited liver metastasis in mice. Together with the in
vitro experiments, we concluded that thrombin
plays
an important role in the progression of lung cancer.
r-hirudin and DTIP inhibitthrombin-promotedcell
migration, invasion and angiogenesis.
To further explore the role of thrombin in the lung cancer, we used
exogenous thrombin to treat NSCLC cell lines in vitro. DTIP and
r-hirudin which are direct thrombin inhibitors, were developed by our
group.
A549,
Lewis
(Fig.
3A) and 95D cells
(supplementary
Fig. S2A) incubated with 10 nmol/L thrombin displayed a remarkable
promotion
in the ability to migrate into the blank space
compared
with the normal control (NC) group. However,
r-hirudin
and DTIP blocked thrombin-enhanced wound-closure capability of NSCNC
cells. Transwell assay results showed that thrombin-driven migration was
inhibited by pre-treatment with
r-hirudin
and DTIP in A549, Lewis (Fig. 3B) and 95D cells
(supplementary
Fig. S2B). Rho GTPases are well known as regulators of actin
cytoskeletal organization and cell motility. Therefore, we examined the
effect of r-hirudin and DTIP on the activation of RhoA and the status of
actin filament organization. Our findings indicated that r-hirudin and
DTIP could suppress the activation of RhoA
in
thrombin-stimulated A549 cells
(Fig.
3C). As illustrated in Fig. 3D, thrombin significantly enhanced the
fluorescence
intensity of polymerized actin (F-actin) compared with the NS groups.
However, r-hirudin and DTIP could decrease the fluorescence intensity in
thrombin-stimulated cells. The appearance of membrane ruffles and the
formation of lamellipodia were also decreased in
r-hirudin-
and DTIP-treated cells. (Fig. 3E, 3F and supplementary Fig. S2C). These
data demonstrated that r-hirudin and DTIP decreased the amount of
F-actin and the formation of lamellipodia in
thrombin-stimulated
NSCLC cells, which consequently leads to decreased cell motility and
migration ability.
In previous studies, we found thrombin deficiency could reduce the
expression of MMP9 and IL6. Furthermore, the expression of MMP9 and IL6
also could be increased by exogenous thrombin, which could be inhibited
by r-hirudin and DTIP (Fig. 3G and supplementary Fig. S3E-G). RhoA could
be activated by thrombin (Fig. 3C). CCG, an inhibitor of RhoA, could
inhibit thrombin-induced expression of MMP9 and IL6. LPA, an activator
of RhoA, could prevent the inhibition induced by r-hirudin and DTIP
(Fig. 3G and supplementary Fig. S3E-G).
Thrombin has been reported to activate NF-κB signaling in human pleural
mesothelial. The effect of r-hirudin and DTIP on the NF-κB pathway in
thrombin-stimulated NSCLC cells was analyzed. The results indicated that
thrombin could activate NF-κB signaling in NSCLC cells. Compared with
the thrombin-treated group, r-hirudin and DTIP exhibited diminished IκBα
and p65 phosphorylation, suggesting that r-hirudin and DTIP can inhibit
thrombin-induced NF-κB activation. CCG could inhibit the
thrombin-induced NF-κB activation, while RhoA or NF-κB activatiors (LPA
and LPS) could prevent the inhibition induced by r-hirudin and DTIP
(supplementary Fig. S3A-D). The results indicated that thrombin could
activate NF-κB signaling via RhoA in NSCLC cells.
Studies have shown that NF-κB pathway activation upregulates the
expression of cell adhesion molecules and inflammatory cytokines.
We
also found RhoA and NF-κB inhibitor could inhibit thrombin-induced
expression of MMP9 and IL6, RhoA and NF-κB activator could prevent the
inhibition induced by r-hirudin and DTIP (Fig. 3G and supplementary Fig.
S3E-G), suggesting that thrombin can regulate the expression of MMP9 and
IL6 via RhoA and NF-κB pathway.
Thrombin is known to promote the release VEGF and
induce
angiogenesis. Hence, we examined the effects of r-hirudin and DTIP on
angiogenesis using a tube formation assay. After thrombin treatment, the
tubule formation was promoted, r-hirudin and DTIP significantly inhibit
thrombin-induced tube formation (Fig. 3H). These results demonstrated
that r-hirudin and DTIP possessed an anti-angiogenic potential.
r-hirudin and DTIP exert anti-invasive andanti-metastatic
abilities in a mouse lung cancer model.
Our aforementioned results suggest
anti-metastatic
and anti-angiogenic activity of
r-hirudin
and DTIP in vitro. We further confirmed the effects in vivo.
In
orthotopic lung tumor model, DTIP could improve the survival time of
mice compared with the control group, (Fig. 4A), and mice in r-hirudin
or DTIP-treated groups had smaller tumor burden (Fig. 4B), had fewer
mice with liver metastases (Fig. 4C).
Murine models of experimental metastasis have been used frequently to
investigate the effects of anti-haemostatic agents on cancer metastasis.
Although
such artificial models do not encompass the entire metastatic process,
they remain useful for
‘proof-of-concept’-experiments,
focusing
on the
haematogenous
phase of tumor dissemination (Mammadova-Bach et al., 2020; Sjoberg et
al., 2019; Vuong et al.,
2019).
Gross examination of the lungs harvested from
r-hirudin-
or DTIP-treated mice revealed a median of 21 (n=12) or 20 (n=12) surface
pulmonary metastases per animal. In contrast, the lungs harvested from
normal
saline treated mice (n=16) had confluent metastases that were too
numerous to count and were clearly enlarged (Fig. 4D, 4E). Micro-PET
scan and histologic analyses revealed scattered small foci of tumor
tissue within the lungs harvested from r-hirudin- or DTIP-treated mice,
while the lungs harvested from
normal
saline-treated mice were nearly completely effaced by tumor tissue
(Fig.
4F,
4G).
We found the number of mice with
tumor
cells colonized in the liver was largely reduced in the r-hiruin and
DTIP group compared with the
normal
saline group (Fig. 4I, 4J). It is important to note 78% of control mice
(n=9) were
dead
at 24 days, with all dead at 32 days, whereas 25% of r-hirudin-treated
mice (n=12) and 30% of DTIP-treated mice (n=10) were dead at 24 days
(Fig. 4H).
We
also examined spontaneous metastasis through subcutaneous inoculation of
tumor cells in mice, which involves a more
comprehensive
process.
Treatment
of r-hirudin and DTIP for one week inhibited tumor growth slightly
(Fig.
5A, 5B).
Six
of the 9 tumors analyzed from normal saline-treated mice showed signs of
panniculus invasion, whereas only
2
of 9 tumors from r-hirudin-treated mice and 2 of 10 tumors from
DTIP-treated mice had any noticeable signs of
panniculus
invasion (Fig. 5C,
5D).
And we also
administered
1.0 mg/kg DTIP or 0.5 mg/kg r-hirudin for 21 consecutive days after one
week of the injection of LLC cells. r-hirudin and DTIP significantly
inhibited tumor growth
(Fig.
5E-G).
The
number of mice with lung and liver
metastases
was largely reduced in r-hirudin or DTIP treated groups (Fig. 5H-K).
Tumor angiogenesis was assessed using IHC analysis for CD31. The
r-hirudin or DTIP treated groups showed a significant reduction of
CD31-positive microvessels versus controls (Fig. 5L).
We performed
immunohistochemical
analysis on tumor samples to determine the expression levels of MMP9 and
IL6. There is decreased
expression
of
MMP9
and IL6 after treatment with r-hirudin and DTIP compared with normal
saline-treated mice
(Fig.
5M). We also examined phosphorylation levels of p65 and the key
downstream signaling molecules intratumorally. We observed a marked
inhibition
of phospho-p65,
phospho-Erk,
phospho-STAT3, and phospho-Akt levels in the r-hirudin- and DTIP-treated
groups (Fig. 5N).
Furthermore,
we did not find increased bleeding after administration of DTIP, slight
subcutaneous hemorrhage was observed after r-hirudin administration for
three weeks continuously (data are not shown). These results show that
DTIP, a direct thrombin inhibitor, could be extended to anti-cancer
therapy.
PAR-1 is a major determinant in thrombin-promotedmetastatic
of lung cancer.
Thrombin is the main activator of PAR-1 (Vu, Hung, Wheaton & Coughlin,
1991). Overexpression of PAR-1 has been detected in various types of
cancers, including ovarian (Grisaru-Granovsky, Salah, Maoz, Pruss,
Beller & Bar-Shavit, 2005), breast cancer (Boire, Covic, Agarwal,
Jacques, Sherifl & Kuliopulos, 2005), lung cancer, prostate cancer
(Black et al., 2007), and melanoma. Our previous experimental results
also showed PAR-1 was highly expressed in human and mouse tumors
compared with normal lung tissues (supplementary Fig. S4). However,
we
did not find an obvious relationship between the PAR-1 expression levels
of tumors and
clinical
variables, such as the
stage
of NSCLC differentiation status and disease
progression
(supplementary Table 1).
To
observe the role of PAR-1 in thrombin-mediated invasion and metastasis
more clearly, we constructed A549PAR-1-/- and
LLCPar-1-/- cells.
PAR-1
depletion almost completely abrogated thrombin-promoted cell migration,
and the effect of r-hirudin and DTIP on thrombin-induced migration and
invasion was abolished (Fig. 6A-D). PAR-1 is a G protein coupled
receptor and has been shown to induce cellular invasion through
RhoA-dependent signaling. After depleting PAR-1, the activation of RhoA
was inhibited and the ability of thrombin to activate RhoA was also
inhibited (Fig. 6E). Similar responses were also observed via
immunofluorescence staining of F-actin, as
PAR-1
depletion decreased the formation of membrane ruffles
(Fig.
6F). These data suggest thrombin-enhanced cell motility and migration
can be completely abrogated by PAR-1 depletion in vitro. PAR-1
deficiency
exhibited diminished
IκBα
phosphorylation and p65 phosphorylation (supplementary Fig. S5A).
Importantly, thrombin-driven NF-κB activation were inhibited by
pre-treatment with the specific PAR-1 inhibitor ML161 (supplementary
Fig. S5A). LPA or LPS could rescue the activation of NF-κB, but thrombin
could not. r-hirudin and DTIP could not inhibit NF-κB activation induced
by LPA or LPS (supplementary Fig. S5B,5C), suggesting r-hirudin and DTIP
inhibit thrombin-induced RhoA and NF-κB activation via PAR-1 signaling.
We also found PAR-1 deficiency exhibited deceased expression of MMP9 and
IL6. Besides thrombin, LPA or LPS could increase MMP9 and IL6 expression
in PAR-1 deficient cells. r-hirudin and DTIP could not inhibit MMP9 and
IL6 expression induced by LPA or LPS (supplementary Fig. S6), suggesting
r-hirudin and DTIP inhibit thrombin-induced MMP9 and IL6 expression
through RhoA and NF-κB activation via PAR-1 signaling.
To further analyze the effects of PAR-1 on lung cancer growth and
metastasis, we established lung cancer model in mice using
LLC
cells infected by gRNA-PAR-1
lentivirus
(Par-1-/- group) or LV-negative control
(NC,
vehicle group). In orthotopic lung tumor model, PAR-1 deficiency could
markedly increase the survival rate and inhibit tumor growth in lung
(Fig. 6G-I).
In the metastatic colonization model, our results confirmed PAR-1
deficient
lung cancer cells lead to less lung metastatic nodes than control cells
(supplementary Fig. S7A, 7B), as well as significantly decreased signal
intensity in the lungs, as seen on micro-PET scans (supplementary Fig.
S7C).
In
addition, PAR-1 deficiency could markedly increase the survival rate
(supplementary Fig. S7D).
In subcutaneous tumors,
PAR-1
deficient group were significantly smaller than control group
(Fig.
6J, 6K).
The
lung metastases and liver metastases were both largely reduced in PAR-1
deficient group (supplementary Fig. S7E). In addition, based on the
results of CD31 staining, we confirmed that CD31-positive microvessels
decreased in
the
PAR-1
deficient
tumors
(Fig.
6L). We also found the levels of phospho-p65, phospho-Erk,
phospho-STAT3, and phospho-Akt were reduced in PAR-1 deficient tumors
(supplementary
Fig. S7F). Together with the in vitro experiments, we concluded that
PAR-1 plays an important role in the thrombin-induced progression of
lung cancer.
DTIP
potentiateschemotherapy-induced
growth and metastasis inhibition and inhibits chemotherapeutic drug
tolerance of NSCLC in mice.
Chemotherapy
has been commonly prescribed in the treatment of patients with NSCLC,
however, its benefits are limited due to a low response rate or acquired
tumor resistance. Arnold et. al have shown that PAR-1 in the tumor
induces the chemo-resistance of cancer (Queiroz et al., 2014).
Meanwhile, chemotherapy such as gemcitabine,
cisplatin,
and paclitaxel are associated with a significant increase in the risk of
arterial thromboembolic events (Zaborowska-Szmit, Krzakowski, Kowalski
& Szmit, 2020). we hypothesized that DTIP could potentiate
chemotherapy-induced inhibition of tumor progression.
When
combination
DTIP and gemcitabine, the
tumor
volume and tumor weight
in
the combination treatment group was significantly smaller than that in
the
groups
administered DTIP alone or
gemcitabine
alone
(Fig.
7A-C). The number of mice with lung metastases was largely reduced in
the combination treatment group
(Fig.
7D,
7E).
We
also counted the survival rates of mice in different groups. It is
important to note 85% of control mice (n=7) were dead at 60 days, 57%
of r-hirudin-treated mice (n=7) and 50% of
gemcitabine
-treated mice
(n=8)
were dead at 60 days, whereas, 16% combination -treated mice (n=6) were
dead at 60 days
(Fig.
7F). Paclitaxel could not significantly inhibit the growth of LLC in
vivo. However, when combined with DTIP, the growth (Fig. 7G-I) and
metastasis (Fig. 7J, 7K) of LLC were significantly inhibited.
Combination of DTIP and cisplatin had a smaller tumor volume
(supplementary Fig. S8A-C), but there was no significant difference in
metastasis and survival time of mice compared with the group
administered cisplatin alone (supplementary Fig. S8D, 8E). In addition,
we evaluated the chemotherapy effects in thrombin deficient NSCLC mouse
models. We found gemcitabine or paclitaxel treated mice in thrombin
deficient group had smaller tumors (supplementary Fig. S9A, 9C) and
longer survival time (supplementary Fig. S9B, 9D) compared with control
group treated with gemcitabine or paclitaxel. These results indicated
that combination therapy of DTIP and chemotherapy might achieve a better
therapeutic effect.