DDIT4 Facilitates Lymph Node Metastasis via the Activation of NF-κB Pathway and Epithelial-Mesenchymal Transition

概要

主論文の要旨

DDIT4 Facilitates Lymph Node Metastasis via the
Activation of NF-κB Pathway and
Epithelial-Mesenchymal Transition
DDIT4はNF-κBシグナル伝達経路の活性化と上皮間葉転換を介して
子宮頸癌のリンパ節転移を促進する

名古屋大学大学院医学系研究科
発育・加齢医学講座

総合医学専攻

産婦人科学

（指導：梶山 広明 教授）
林 欣欣

【Introduction】
Cervical cancer ranks as the fourth most frequently diagnosed cancer and the fourth leading
cause of cancer death in women globally. A major challenge in the treatment of patients with
cervical cancer is metastasis, as the outcome of patients with metastatic cervical cancer is poor.
Recent studies have shown that a morphological transformation called epithelial-mesenchymal
transition (EMT) and hypoxia in the tumor microenvironment are associated with malignant
transformation in cervical cancer cells. An EMT plays an important role in tumor progression
and is related to many features of cancer. Clinical data have demonstrated that an EMT is
closely related to poor prognoses in cervical cancer patients. A prominent feature of clinically
advanced cervical cancers is hypoxia, which is a therapeutic and prognostic factor that is
associated with metastasis, resistance to chemotherapy and radiotherapy, and poor patient
survival. A central coordinator of the hypoxic cellular response is hypoxia-inducible factor1alpha (HIF-1α), a master transcription factor that regulates the expression of a growing list
of downstream targets. Hypoxia and HIF-1α influence multiple steps in the metastatic cascade,
including invasion and migration, intravasation and extravasation, and the establishment of the
premetastatic niche, as well as survival and growth at the distant site(s). The mechanism
underlying how hypoxia induces metastasis in cervical cancer has not been established. We
conducted the present study to identify a novel metastasis-promoting molecule by the analysis
of multiple databases, and found that DDIT4 (DNA-damage-inducible transcript 4), also
known as REDD1/RTP801/Dig1, a hypoxia-inducible gene, is closely associated with
metastasis and an EMT in cervical cancer patients. DDIT4 was initially identified as a hypoxiaregulated HIF-1 target gene. Its strong up-regulation by hypoxia was detected both in vitro and
in an animal model of ischemic stroke. High DDIT4 expression is associated with a poor
prognosis in multiple cancer types. However, the clinical significance and biological role of
DDIT4 in cervical cancer remain to be elucidated.
【Purpose】
This study was aimed to identify a novel metastasis-promoting molecule and elucidate its
functional and prognostic roles in cervical cancer.
【Methods】
DDIT4 (DNA-damage-inducible transcript 4), a hypoxia-inducible gene, was identified by
analyzing multiple microarray databases. The correlation between DDIT4 expression in
immunohistochemistry and clinicopathological characteristics in the public database and our
cohort was evaluated by statistical analysis. Transwell ® assay and wound-healing assay to
determine cell migration and invasion was performed. DDIT4 was knocked down using siRNA
or lentiviral vectors. The potential downstream pathways of DDIT4 were explored and verified
by a gene set enrichment analysis and western blotting. The in vivo metastatic capability was

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determined with the use of an intraperitoneal-injection mouse model.
【Results】
In analysis of public database, DDIT4 is upregulated in cervical cancer cells (Fig. 1A). A
high level of DDIT4 expression significantly correlated with a poor survival rate in cervical
cancer patients (p=0.00385) (Fig. 1B). The results of univariate and multivariate analyses
demonstrated that DDIT4 is an independent prognostic factor for patients at the early stage of
cervical cancer (p<0.05) (Fig. 1C). In analysis of our cohort, DDIT4 expression in early-stage
cervical cancer was correlated with metastatic potential such as lymphovascular invasion and
lymph node metastasis (Table 1). The expression of HIF-1α was strongly associated with the
expression of DDIT4 in cervical cancer tissues (Fig. 2A, 2B), and expression of DDIT4 might
reflect hypoxic conditions in cervical cancer cell (Fig. 2C). We investigated the expression
patterns of DDIT4 in the human uterine cervical cancer cell lines, HeLa, SiHa, CaSki, QGU
and TCS, and found that in all of the cell lines examined, DDIT4 was constitutively expressed
(Fig. 3A). Among the cell lines we used, mainly HeLa and CaSki were used for the subsequent
experiments, because these two cell lines highly expressed DDIT4. We suppressed the
expression of DDIT4 by DDIT4-specific siRNAs (Fig. 3B). The knockdown of DDIT4
attenuated the migration and invasion activity of tumor cells in vitro (FIG. 3C, 3D), and
reduced the expression of epithelial–mesenchymal transition (EMT) related proteins (FIG. 3E,
3F). DDIT4 activated the nuclear NF-κB pathway, at least partially via IKKα-dependent
regulation (FIG. 4). DDIT4 also promoted tumor progression in the mouse model (FIG. 5).
【Discussion】
The majority of the existing studies on DDIT4 have focused on its role in metabolism. In
the present study, we extended the investigation to the effects of DDIT4 in cervical cancer
metastasis. DDIT4 is overexpressed in cervical cancer, and its expression indicates an
unfavorable clinical outcome. Our present investigations indicate that DDIT4 overexpression
encourages the invasion and migration of cervical cancer cells. We observed that the
overexpression of DDIT4 promoted cell migration invasion and an EMT, and that it activated
the nuclear NF-κB pathway. Since a hypoxic condition the induced expression of DDIT4,
which is regulated by the activation of HIF-1α, we can draw the conclusion that DDIT4 plays
an important role in hypoxia that induces metastasis. We observed herein that the knockdown
of DDIT4 induced the expression of E-cadherin and suppressed the expression of vimentin
(FIG. 3E), indicating that DDIT4 is a promoter of the EMT. The EMT plays an important role
in cervical cancer progression and metastasis. It thus appears that DDIT4 induced cervical
cancer metastasis by promoting the EMT. The Western blotting results verified that DDIT4
induced the expressions of Ser 65 and IKKα and activated the nuclear NF-κB pathway (FIG.
4). Inhibitors of nuclear factor κB kinase (i.e., IKKs), mainly IKKα and IKKβ, are upstream

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regulators of the NF-κB pathway. IKKα activates p65 by phosphorylating p65. Our present
findings revealed for the first time that DDIT4 directly activates the NF-κB pathway through
IKKα in cervical cancer cells. Our speculate that DDIT4 was induced by hypoxia and promoted
metastasis through the EMT and the NF-kB pathway. Clinically, DDIT4 is upregulated in
cervical cancer tissues. A high level of DDIT4 independently indicated a poor prognosis in the
cervical cancer patients in the TCGA database. However, this finding was different in our
clinical specimens; the small sample size might be the reason for this. However, DDIT4 is
significantly correlated with metastasis. This was further demonstrated by the results obtained
with our in vivo model (FIG. 5). Importantly, DDIT4 was also associated with lymph node
metastasis in the model (Table 1), and this finding could provide the focus of future studies.
【Conclusion】
Our results indicates that DDIT4 can be a prognostic indicator in cervical cancer and
promote lymph node metastasis, augmenting malignancy via the EMT and NF-kB pathways.

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