ECD promotes gastric cancer metastasis by blocking E3 ligase ZFP91-mediated hnRNP F ubiquitination and degradation
Xu et al. Cell Death and Disease
ECD promotes gastric cancer metastasis by blocking E3 ligase ZFP91-mediated hnRNP F ubiquitination and degradation
Song-Hui Xu 0 1 2 3
Song Zhu 0 1 2 3
Yanjie Wang 1 3
Jin-Zhou Huang 1 3
Min Chen 0 1 2 3
Qing-Xia Wu 1 3
Yu-Tian He 1 3
De Chen 0 1 2 3
Guang-Rong Yan 0 1 2 3
0 Key Laboratory of Protein Modification and Degradation, Guangzhou Medical University , Guangzhou 510150 , China Edited by M Agostini
1 Biomedicine Research Center, The Third Affiliated Hospital of Guangzhou Medical University , Guangzhou 510150 , China
2 Key Laboratory of Protein Modification and Degradation, Guangzhou Medical University , Guangzhou 510150 , China
3 Biomedicine Research Center, The Third Affiliated Hospital of Guangzhou Medical University , Guangzhou 510150 , China
The human ortholog of the Drosophila ecdysoneless gene (ECD) is required for embryonic development and cell-cycle progression; however, its role in cancer progression and metastasis remains unclear. Here, we found that ECD is frequently overexpressed in gastric cancer (GC), especially in metastatic GC, and is correlated with poor clinical outcomes in GC patients. Silencing ECD inhibited GC migration and invasion in vitro and metastasis in vivo, while ECD overexpression promoted GC migration and invasion. ECD promoted GC invasion and metastasis by protecting hnRNP F from ubiquitination and degradation. We identified ZFP91 as the E3 ubiquitin ligase that is responsible for hnRNP F ubiquitination at Lys 185 and proteasomal degradation. ECD competitively bound to hnRNP F via the N-terminal STG1 domain (13-383aa), preventing hnRNP F from interacting with ZFP91, thus preventing ZFP91-mediated hnRNP F ubiquitination and proteasomal degradation. Collectively, our findings indicate that ECD promotes cancer invasion and metastasis by preventing E3 ligase ZFP91-mediated hnRNP F ubiquitination and degradation, suggesting that ECD may be a marker for poor prognosis and a potential therapeutic target for GC patients.
Gastric cancer (GC) is a prevalent malignancy in East
Asian countries, including China, and is the second
leading cause of cancer-related mortality worldwide, with
an overall 5-year survival rate of less than 25%1,2. Most
GCs are diagnosed clinically at an advanced disease stage
and thus present with distant metastases, which are the
most important cause of cancer-associated death in GC
patients. Although surgical resection is considered the
gold standard for treating GC patients, GC patient
prognosis remains poor due to the high incidence of tumor
recurrence and distant metastasis. Conventional
chemotherapy has limited effects on GC, especially
metastatic GC. Targeted small molecule or antibody
therapies designed to inhibit a specific oncogene are
promising therapeutic strategies. Anti-HER2-targeted
antibody therapies improve the overall survival of
HER2-positive GC patients when combined with
chemotherapy; however, HER2-positive patients comprise
only 7–17% of GC patients. Therefore, new therapeutic
targets are urgently needed.
The ecdysoneless (ECD) gene was originally named by
authors studying Drosophila melanogaster ECD mutants
who exhibited defective development due to reduced
production of the steroid hormone, ecdysone, required for
insect molting3. Subsequent studies showed that the ECD
protein is required for cell-autonomous processes in
Drosophila development and oogenesis4. The human ECD
homolog was initially identified in a complementation
assay conducted to rescue yeast mutants lacking the
glycolysis regulation 2 (Gcr2) gene5. ECD gene deletion in
mouse embryonic fibroblasts led to cell-cycle arrest at the
G1/S checkpoint, suggesting ECD is a novel cell-cycle
regulator4,6. ECD is overexpressed in pancreatic and
HER2/ErbB2-overexpressing breast cancers7,8. Our
previous studies showed that ACK1 promotes GC metastasis
through the AKT-POU2F1-ECD pathway and that ECD is
a potential key downstream effector of ACK11,9. However,
the roles and molecular mechanisms of ECD in cancer
progression and metastasis remain unknown.
hnRNP F belongs to the hnRNP family, a large family of
RNA-binding proteins that regulate multiple aspects of
nucleic acid metabolism, including alternative splicing,
transcription, translation, and mRNA stabilization10.
hnRNP expression is altered in many cancers10,11, and
these proteins are crucial in cancer cell proliferation,
invasion, and metastasis10,12–15. hnRNP F/H regulate
alternative splicing of the apoptotic regulator, Bcl-x, and
the tumor-associated NADH oxidase, ENOX216–18.
hnRNP F is a potential marker for colorectal cancer
progression19; however, the regulatory mechanism of
hnRNP F expression upregulation in cancers remains
Ubiquitination is a well-studied post-translational
modification involved in proteasomal degradation,
protein–protein interaction, protein trafficking, and
protein activity. Protein ubiquitination is mediated by three
enzyme families (E1, E2, and E3). Ubiquitination system
activity depends on E3 ubiquitin ligase specificity20–22. To
date, a direct connection between hnRNP F and the
ubiquitination pathways remains unobserved, as an hnRNP
F-specific E3 ligase that can bind to hnRNP F and induce
ubiquitination and proteasomal degradation of hnRNP F
has not been identified.
In this study, we found that ECD was overexpressed in
GC, especially in metastatic GC, and ECD promotes GC
invasion and metastasis by stabilizing hnRNP F. We
further found that ZFP91 is the E3 ligase responsible for
hnRNP F ubiquitination at Lys 185 and degradation. ECD
blocks the interaction between ZFP91 and hnRNP F and
the subsequent ubiquitination- and degradation-inducing
effects of ZFP91 on hnRNP F by competitively binding to
hnRNP F. Our findings indicate that ECD facilitates
cancer migration and invasion by stabilizing hnRNP F,
and ECD may be used as a novel prognostic GC
biomarker, as well as an anti-cancer therapeutic target.
ECD overexpression is correlated with aggressive GC phenotypes
To investigate the role of ECD in gastric progression, we
analyzed ECD protein levels in six pairs of primary GC
tissue samples and matched adjacent non-tumoral gastric
tissue (N) samples. We found elevated ECD protein levels
in all GC tissue samples compared to those in the N
samples (Fig. 1a). We also investigated ECD mRNA
expression in gastric mucosal tissues and GC tissues using
three microarray gene expression datasets deposited in
the Oncomine database. ECD mRNA levels were higher in
gastric intestinal-type adenocarcinoma and gastric
adenocarcinoma tissues than in gastric mucosal tissues
(Fig. 1b), indicating that ECD expression is upregulated
To investigate the correlation between ECD levels and
prognosis, we performed an extensive tissue microarray
analysis of 186 GC tissue samples and 154 adjacent
nontumoral gastric tissue (N) samples (including 149 GC
tissue pairs and matched non-tumoral gastric tissues)
using an immunohistochemical (IHC) assay (Fig. 1c). We
detected higher ECD levels in GC tissues than in N tissues
(Fig. 1d), as well as higher ECD levels in metastatic GC
tissues than in non-metastatic GC tissues (Fig. 1e).
ECD overexpression was positively associated with pT
status, pN status, lymph node metastasis, more advanced
histological grades and clinical stage in GC (Table 1). GC
patients with high ECD levels were at higher risk for
cancer-related death than those with low ECD levels
(Fig. 1f, g). The mean overall survival time for GC patients
with high ECD levels was 19.5 months, while for GC
patients with low ECD levels, it was 45.5 months (p =
0.0005, log-rank test). Therefore, ECD overexpression was
correlated with poor prognosis in GC patients.
ECD promotes GC invasion and metastasis
To investigate the role of ECD in GC invasion and
metastasis, we either silenced or overexpressed ECD in
two GC cell line models. ECD silencing suppressed GC
cell migration and invasion in SGC-7901 and MGC-803
cells (Fig. 2a), while ECD overexpression promoted GC
cell migration and invasion in those cells (Fig. 2b).
Smaller metastatic nodules developed in mouse lungs
after injection with luciferase-tagged SGC-7901 cells with
stable silencing of ECD expression than those in mouse
lungs after injection with SGC-7901 cells (Fig. 2c). Smaller
metastatic lung nodules were also confirmed by
histological analysis (Fig. 2d). Collectively, these results indicate
bECD expression score is 0–5.
cECD expression score is 6–7.
dAll case number is less than 186 because the clinicopathological information of
some cases is absent or clear.
that ECD promotes GC cell migration and invasion
in vitro and metastasis in vivo.
ECD interacts with hnRNP F via the SGT1 domain
To investigate the mechanism by which ECD promotes
cancer invasion and metastasis, we identified the proteins
that interact with ECD by performing Co-IP and mass
spectrometry analyses (Fig. 3a). We identified that hnRNP
F might interact with ECD. We further confirmed that
ECD interacts with hnRNP F (Fig. 3b).
ECD consists of a conserved SGT1 domain and
transactivation region5. To determine which domains interact
with hnRNP F, we generated truncated ECD constructs
with an N-terminal Flag tag (Fig. 3c). When these
constructs were co-expressed with HA-hnRNP F in cells, only
the constructs containing the ECD N-terminal STG1
domain (13-383aa), but no other ECD regions, could
interact with hnRNP F, indicating that the N-terminal
STG1 domain is essential for hnRNP F binding (Fig. 3d).
ECD increases hnRNP F protein levels by inhibiting hnRNP
F polyubiquitination and degradation
How does ECD affect hnRNP F given that ECD can bind
to hnRNP F? To address this, we investigated the effects
of ECD on hnRNP F protein and mRNA levels. ECD
silencing decreased hnRNP F protein levels, but not
hnRNP F mRNA levels (Fig. 4a, b), while ECD
overexpression increased hnRNP F protein levels, but not
hnRNP F mRNA levels, in a dose-dependent manner
(Fig. 4c, d), indicating that ECD upregulated hnRNP F
protein levels at the post-transcriptional level.
Because ECD may upregulate hnRNP F protein levels,
and the ubiquitination/proteasome pathway is the
quickest known mechanism through which proteins are
irreversibly degraded20,23,24, we surmised that ECD
upregulated hnRNP F by preventing its ubiquitination.
To test this hypothesis, we determined the effect of
ECD overexpression on the half-life of hnRNP F. As
shown in Fig. 4e, ECD overexpression resulted in a longer
hnRNP F half-life. We also performed an in vivo
ubiquitination assay, which showed that silencing ECD
in the presence of MG132 (a specific proteasome
inhibitor) increased hnRNP F polyubiquitination levels
(Fig. 4f). Collectively, our results strongly indicate that
ECD inhibits hnRNP F protein polyubiquitination
and proteasomal degradation, thus increasing hnRNP F
Furthermore, we investigated the correlation between
ECD and hnRNP F protein levels in GC tissues. We found
that hnRNP F protein levels were upregulated in GC
tissues compared with those in matched N tissues,
consistent with the results of the ECD expression
experiments (Fig. 1a). Extensive tissue microarray analysis
showed that hnRNP F protein levels were higher in GC
tissues than in N tissues (Fig. 4g); however, hnRNP F
mRNA levels were unchanged between GC tissues and N
gastric tissues (Supplementary Fig. S1). ECD protein levels
were also positively correlated with hnRNP F protein
levels in GC tissue samples (R = 0.314, p < 0.0001)
(Fig. 4h). These findings indicate that ECD increases
hnRNP F protein levels by stabilizing hnRNP F.
ECD stimulates GC migration and invasion by stabilizing hnRNP F
Next, we investigated the role of hnRNP F in ECD
functions. We found that silencing of hnRNP F inhibited
GC cell migration and invasion, similar to the effects
induced by ECD knockdown (lane 3 in Supplementary
Fig. S2B). And silencing hnRNP F attenuated the
enhanced migration and invasion induced by ECD
overexpression (Supplementary Fig. S2), indicating that ECD
promotes GC migration and invasion by regulating
hnRNP F stability.
ZFP91 is an E3 ubiquitin ligase responsible for ubiquitinating hnRNP F
As ECD is not an E3 ligase, and the E3 ligase responsible
for hnRNP F ubiquitination remains unknown, we sought
to identify the E3 ligase that regulates hnRNP F protein
ubiquitination and degradation. E3 ligases must bind to
their substrates to facilitate their ubiquitination. Thus, we
identified the proteins that interacted with hnRNP F by
performing Co-IP and a mass spectrometry assay to
identify the E3 ligase responsible for ubiquitinating
hnRNP F. We found that ZFP91 is the potential E3 ligase
because it can bind to hnRNP F (Fig. 5a). We further
confirmed that ZFP91 interacts with hnRNP F (Fig. 5b, c),
thus suggesting that ZFP91 may be the E3 ligase
responsible for polyubiquitinating hnRNP F.
To determine whether ZFP91 is the E3 ligase
responsible for hnRNP F ubiquitination, we investigated the
effects of ZFP91 on hnRNP F mRNA and protein levels,
half-life and ubiquitination. We found that silencing
ZFP91 increased hnRNP F protein levels, but not hnRNP
F mRNA levels, while ectopically expressing ZFP91
decreased hnRNP F protein levels, but not hnRNP F
mRNA levels (Fig. 5d, Supplementary Fig. S3). Moreover,
ZFP91 overexpression resulted in a shorter hnRNP F
protein half-life (Fig. 5e), and our in vivo ubiquitination
assay results showed that ZFP91 overexpression increased
hnRNP F polyubiquitination levels in the presence of the
specific proteasome inhibitor, MG132 (Fig. 5f). This
indicates that ZFP91 is the E3 ligase responsible for
ubiquitinating and degrading hnRNP F.
ZFP91 induces hnRNP F ubiquitination at Lys 185
To identify the specific ubiquitination modification
lysine sites in the hnRNP F protein, we searched the
PhosphoSitePlus post-translational modification resource.
Four potential ubiquitination sites at lysine residues were
found in the hnRNP F protein (Supplementary Fig. S4A).
We subsequently generated hnRNP F mutants in which
these lysine residues were replaced with arginine. We
found that ZFP91 overexpression did not change the
protein levels in the hnRNP F mutant with K185R, but it
decreased the protein levels in the hnRNP F mutants with
K87R, K167R and K171R (Fig. 5g). ZFP91 overexpression
also increased the hnRNP F mutant polyubiquitination
levels with K87R, K167R, or K171R, but not in the mutant
with K185R (Fig. 5h). These data indicate that hnRNP F
ubiquitination at Lys 185 was regulated by ZFP91 E3
We further investigated the effects of hnRNP F
ubiquitination at Lys 185 on ZFP91-mediated cancer cell
migration and invasion. We found that ZFP91
overexpression inhibited GC cell migration and invasion, thus
exerting effects that contrast with those of hnRNP F
(Supplementary Fig. S4B). ZFP91 co-expression also
attenuated the enhanced migration and invasion induced
by overexpressing the hnRNP F mutants with K87R,
K167R, or K171R, as ZFP91 polyubiquitinated these
hnRNP F mutants and facilitated their subsequent
degradation by the proteasome; however, ZFP91
coexpression did not block the enhanced migration and
invasion induced by overexpressing the hnRNP F mutant
with K185R because this protein was not
polyubiquitinated and was degraded by ZFP91
(Supplementary Fig. S4B). Collectively, our results indicate that ZFP91
polyubiquitinated hnRNP F at Lys 185.
ECD blocks ZFP91 from binding to hnRNP F
We further investigated, and subsequently found, an
interaction between ECD and ZFP91 (Fig. 6a, b). The
constructs containing the ECD N-terminal STG1 domain
(13-383aa), but not other ECD regions, retained
interactions with ZFP91 (Fig. 6c), suggesting that the same ECD
domain that bound to hnRNP F interacted with ZFP91.
Because ECD binds to ZFP91 and hnRNP F, and ZFP91
binds to hnRNP F, we surmised that ECD inhibits the
interaction between ZFP91 and hnRNP F. We
cotransfected ZFP91, hnRNP F, and ECD plasmids into
HeLa cells and assessed the protein interactions. We
found that ECD overexpression dose-dependently
blocked the ZFP91 and hnRNP F interactions (Fig. 6d,
e). Furthermore, we found that the constructs containing
the ECD N-terminal STG1 domain (13-383aa), but not
other ECD regions, blocked the ZFP91 and hnRNP F
interactions (Fig. 6f). This indicates that ECD blocked
ZFP91 from binding to hnRNP F through the N-terminal
ECD blocks ZFP91-mediated hnRNP F ubiquitination and degradation
Because ZFP91 is an E3 ubiquitin ligase that acts on
hnRNP F, and ECD blocks the interaction between ZFP91
with hnRNP F and inhibits hnRNP F degradation, we
surmised that ECD blocks ZFP91-mediated hnRNP F
ubiquitination and degradation. We co-transfected ZFP91,
hnRNP F, and ECD plasmids into HeLa cells and
investigated hnRNP F protein and ubiquitination levels. ECD
reversed the decreased hnRNP F protein levels induced by
ZFP91 overexpression (Fig. 7a). In addition, ECD
dosedependently blocked the enhanced hnRNP F
ubiquitination levels induced by ZFP91 overexpression (Fig. 7b, c).
We further investigated whether the ECD N-terminal
STG1 domain can block ZFP91-mediated hnRNP F
ubiquitination, as this domain blocked the interaction
between ZFP91 and hnRNP F. The mutants containing
the ECD N-terminal STG1 domain exerted effects similar
to wild-type ECD and blocked the increased hnRNP F
polyubiquitination levels induced by ZFP91, while the
mutants containing other ECD regions did not (Fig. 7d).
This indicates that ECD blocks ZFP91-mediated hnRNP F
ubiquitination and degradation through the N-terminal
We found that ECD induces cancer invasion and
metastasis, and we elucidated the novel mechanism
underlying ECD’s effect on cancer progression.
Specifically, we found that ECD stabilizes hnRNP F, by blocking
the interaction between ZFP91 and hnRNP F and the
subsequent ubiquitination and degradation of hnRNP F
by ZFP91. ZFP91 is an E3 ubiquitin ligase that
ubiquitinates hnRNP F at Lys 185.
Initial studies demonstrated that the Drosophila ECD is
required for embryonic development4,78,25. Here, we
demonstrated that human ECD homolog levels were
significantly increased in GC tissues, especially in
metastatic GC tissues, compared with those in adjacent
nontumoral gastric tissues. ECD mRNA levels were also
increased in GC tissues compared with those in the
gastric mucosa tissues in three HCC datasets deposited in the
Oncomine database. These results indicated that ECD
upregulation in GC was induced at the transcriptional
level, consistent with our previous study in which we
observed that ACK1 stimulated the transcription factor
POU2F1 to induce ECD transcription1. ECD
overexpression was significantly associated with aggressive GC
phenotypes, as GC patients with high ECD levels
exhibited higher death rates and shorter survival times
than GC patients with low ECD levels. Our findings
suggest that ECD may be a novel independent prognostic
factor in GC patients. Silencing ECD significantly
suppressed GC metastasis and invasion in vitro and in vivo,
indicating that ECD may be a novel therapeutic target
Previous studies reported that ECD promotes cell
proliferation by regulating RB/E2F pathway-dependent
cellcycle progression and GLUT4-dependent glycolysis in
breast and pancreatic cancer, respectively4,8. Here, we
elucidated a novel mechanism underlying the effects of
ECD on GC invasion and metastasis. We showed that
ECD promoted GC metastasis and invasion by stabilizing
hnRNP F. We found that ECD bound to hnRNP F to
prevent E3 ligase ZFP91 from interacting with hnRNP F,
thus blocking ZFP91-mediated hnRNP F
polyubiquitination at Lys 185 and increasing hnRNP F protein levels to
promote cancer metastasis and invasion. Previous studies
showed that hnRNP F mRNA levels were increased by
OKI-6 in myelinating glia26. In this study, we discovered a
novel mechanism through which hnRNP F is upregulated
by ECD blocking the ubiquitin/proteasome pathway. The
double bands for hnRNP F were detected; the possible
causes include the different isoform of hnRNP F and the
cleaved hnRNP F.
ZFP91 is a novel E3 ligase that activates the
NF-κBinducing kinase (NIK) via Lys63-linked ubiquitination in
the noncanonical NF-κB signaling pathway27. Here, we
noted that in addition to activating substrates via
ubiquitination, ZFP91 also interacted with and promoted the
ubiquitination of hnRNP F at Lys 185, as well as its
subsequent degradation by the proteasome. Here, we report
for the first time that ZFP91 is an E3 ligase that
ubiquitinates and degrades hnRNP F.
In conclusion, we found that ECD is overexpressed in
GC, especially in metastatic GC, and that ECD
overexpression is correlated with a malignant phenotype and
poor prognosis for GC patients. We elucidated the novel
molecular mechanism underlying the effects of ECD in
cancer progression, as we determined that ECD promotes
invasion and metastasis by stabilizing hnRNP F. We
further demonstrated that ECD competitively bound to
hnRNP F through the N-terminal STG1 domain to
prevent ZFP91-mediated hnRNP F ubiquitination and
degradation. We discovered the E3 ligase, ZFP91, which is
responsible for hnRNP F ubiquitination and degradation
at Lys 185. Our findings indicate that ECD may be
a new prognostic factor and potential anti-cancer target
Materials and Methods
Cell culture and tissue samples
HeLa cells were obtained from and authenticated by
ATCC. The GC cell lines, SGC-7901 and MGC-803, were
obtained from and authenticated by isoenzyme assay by
the Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences (Shanghai, China)28. The cells were
cultured as previously described1,9. All cell lines were
treated with Plasmocin and tested with Mycoplasma PCR
Detection Kit (Sigma-Aldrich, USA). Primary GC tissue
samples and matched adjacent non-tumoral gastric tissue
samples were collected at The Third Affiliated Hospital of
Guangzhou Medicine University. Samples with a clear
pathological diagnosis were selected and obtained from
GC patients who had not received preoperative
anticancer treatment. Informed consent was obtained from
each patient who participated in the study, and tissue
sample collection was approved by the Internal Review
and Ethics Boards of The Third Affiliated Hospital of
Guangzhou Medicine University. Tissue microarray chips
containing GC tissue samples and matched adjacent
nontumoral gastric tissue samples along with their
corresponding clinicopathological data were obtained from
Shanghai OUTDO Biotech Co., Ltd. (Shanghai, China).
Immunohistochemistry (IHC) staining assay
IHC staining assays were performed on tissue
microarray chips with anti-ECD and anti-hnRNP F antibodies,
as previously described, with minor modifications29. All
IHC staining results were assessed by two independent
pathologists blinded to the sample origins and the
corresponding patient outcomes. ECD and hnRNP F
expression was evaluated using a previously described
semi-quantitative German scoring system, which assesses
protein expression based on staining intensity and area.
Scores of 0–5 indicated low ECD and hnRNP expression
(low) in the GC tissue, and scores of 6–7 indicated high
ECD and hnRNP expression (high) in the GC tissue.
Migration and invasion assays
In vitro migration and invasion assays were performed
using Transwell chambers, as previously described1.
Western blotting was performed as previously described1.
Experimental in vivo metastasis model
Male NOD-SCID mice (aged 4–5 weeks) were obtained
from Charles River Laboratories in China (Beijing). The
mice were bred and maintained under defined conditions
at the Animal Experiment Center of the College of
Medicine (SPF grade), Jinan University. The mice were
randomized to allocate into experimental groups by the
researchers blinded to the subject outcomes. In vivo
metastatic assays were performed as previously described,
with minor modifications30. Briefly, five NOD-SCID mice
in each experimental group were injected with
luciferaselabeled SGC-7901-Luc-NC or SGC-7901-Luc-ECD
shRNA-transduced cells (2 × 106 cells in 0.2 ml−1 of
PBS) via their tail veins. The resultant metastatic foci in
the lungs were visualized 2 months after tumor
implantation. Animal experiments were approved by the
Laboratory Animal Ethics Committee of Jinan University
and conformed to the legal mandates and national
guidelines for the care and maintenance of laboratory
Cells were transfected with Flag-ECD, Flag-hnRNP F
(Flag-F), HA-ZFP91, or Flag (negative control) vectors
for 48 h. Co-IP was subsequently performed using
antiFlagor HA antibodies (MBL), and the immune complexes
were captured on protein A/G agarose beads (Santa Cruz,
USA) and separated by SDS-PAGE. The SDS-PAGE
gels were stained with silver. Protein expression was
detected by western blotting using the indicated
Protein identification by mass spectrometry
The differential gel bands and their corresponding
negative gel bands were excised and digested using in-gel
trypsin. The extracted peptide mixtures were analyzed
using nano-LC-MS/MS, as previously described31.
Proteins were identified using the Mascot (v2.3.02) program
against the Uniprot human protein database (released
Dec. 2014) using the default settings. Protein scores were
≥40, and unique peptides were ≥2.
In vivo ubiquitination assay
This procedure was performed as previously
described22. Briefly, the cells were co-transfected with the
indicated plasmids for 36 h, then treated with 10 μM
MG132 for 4 h prior to harvesting. The cells were lysed in
RIPA buffer with protease inhibitor cocktail (Roche,
Switzerland). Flag-hnRNP F was immunoprecipitated
using anti-Flag antibodies and protein A/G agarose beads.
Polyubiquitinated hnRNP F was detected using anti-HA
Two-tailed Student’s t-tests or Mann–Whitney U tests
were used for comparisons between two groups. Survival
analysis was performed using the Kaplan–Meier method
and the log-rank test. Statistical analyses were performed
using Prism 5.0 software. Data are presented as the mean
± SEM unless otherwise stated. *p < 0.05 or **p < 0.01 was
considered statistically significant.
This work was supported by the National Natural Science Foundation of China
(81772998, 81672393), the R&D Plan of Guangzhou (201704020115), the
Yangcheng Scholars program from the Ministry of Education of Guangzhou
(1201561583), Innovative Research Team of Ministry of Education of
Guangzhou (1201610015), the R&D Plan of Guangdong (2017A020215096),
and the National Funds of Developing Local Colleges and Universities
Conflict of interest
The authors declare that they have no conflict of interest.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies this paper at https://doi.org/
1. Xu , S. H. et al. ACK1 promotes gastric cancer epithelial-mesenchymal transition and metastasis through AKT-POU2F1-ECD signalling . J. Pathol . 236 , 175 - 185 ( 2015 ).
2. Deng , N. et al. A comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co-occurrence among distinct therapeutic targets . Gut 61 , 673 - 684 ( 2012 ).
3. Garen , A. , Kauvar , L. & Lepesant , J. A. Roles of ecdysone in Drosophila development . Proc. Natl Acad. Sci. USA 74 , 5099 - 5103 ( 1977 ).
4. Kim , J. H. et al. Role of mammalian Ecdysoneless in cell cycle regulation . J. Biol. Chem . 284 , 26402 - 26410 ( 2009 ).
5. Kim , J. H. , Gurumurthy , C. B. , Band , H. & Band , V. Biochemical characterization of human Ecdysoneless reveals a role in transcriptional regulation . Biol. Chem . 391 , 9 - 19 ( 2010 ).
6. Mir , R. A. et al. A novel interaction of ecdysoneless (ECD) protein with R2TP complex component RUVBL1 is required for the functional role of ECD in cell cycle progression . Mol. Cell. Biol . 36 , 886 - 899 ( 2015 ).
7. Zhao , X. et al. Overexpression of a novel cell cycle regulator ecdysoneless in breast cancer: a marker of poor prognosis in HER2/neu-overexpressing breast cancer patients . Breast Cancer Res. Treat . 134 , 171 - 180 ( 2012 ).
8. Dey , P. et al. Overexpression of ecdysoneless in pancreatic cancer and its role in oncogenesis by regulating glycolysis . Clin. Cancer Res . 18 , 6188 - 6198 ( 2012 ).
9. Xu , S. H. et al. Amplification of ACK1 promotes gastric tumorigenesis via ECD-dependent p53 ubiquitination degradation . Oncotarget 8 , 12705 - 12716 ( 2017 ).
10. Geuens , T. , Bouhy , D. & Timmerman , V. The hnRNP family: insights into their role in health and disease . Hum. Genet . 135 , 851 - 867 ( 2016 ).
11. Chang , E. T. , Parekh , P. R. , Yang , Q. , Nguyen , D. M. & Carrier , F. Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells . Oncotarget 7 , 10578 - 10593 ( 2016 ).
12. Wang , F. et al. SPSB1-mediated HnRNP A1 ubiquitylation regulates alternative splicing and cell migration in EGF signaling . Cell. Res . 27 , 540 - 558 ( 2017 ).
13. Tamayo , J. V. , Teramoto , T. , Chatterjee , S. , Hall, T. M. & Gavis , E. R. The Drosophila hnRNP F/H homolog glorund uses two distinct RNA-binding modes to diversify target recognition . Cell Rep . 19 , 150 - 161 ( 2017 ).
14. Hong , X. et al. PTEN antagonises Tcl1/hnRNPK-mediated G6PD pre-mRNA splicing which contributes to hepatocarcinogenesis . Gut 63 , 1635 - 1647 ( 2014 ).
15. Cammas , A. et al. hnRNP A1-mediated translational regulation of the G quadruplex-containing RON receptor tyrosine kinase mRNA linked to tumor progression . Oncotarget 7 , 16793 - 16805 ( 2016 ).
16. Dominguez , C. , Fisette , J. F. , Chabot , B. & Allain , F. H. Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs. Nat . Struct. Mol. Biol . 17 , 853 - 861 ( 2010 ).
17. Garneau , D. , Revil , T. , Fisette , J. F. & Chabot , B. Heterogeneous nuclear ribonucleoprotein F/H proteins modulate the alternative splicing of the apoptotic mediator Bcl-x . J. Biol. Chem . 280 , 22641 - 22650 ( 2005 ).
18. Tang , X. , Kane , V. D. , Morre , D. M. & Morre , D. J. hnRNP F directs formation of an exon 4 minus variant of tumor-associated NADH oxidase (ENOX2) . Mol. Cell . Biochem. 357 , 55 - 63 ( 2011 ).
19. Balasubramani , M. , Day , B. W. , Schoen , R. E. & Getzenberg , R. H. Altered expression and localization of creatine kinase B, heterogeneous nuclear ribonucleoprotein F, and high mobility group box 1 protein in the nuclear matrix associated with colon cancer . Cancer Res . 66 , 763 - 769 ( 2006 ).
20. Scott , D. C. et al. Two distinct types of E3 ligases work in unison to regulate substrate ubiquitylation . Cell 166 , 1198 - 1214 e1124 ( 2016 ).
21. Kong , S. et al. Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas . Proc. Natl Acad. Sci. USA 113 , 10394 - 10399 ( 2016 ).
22. Hong , J. et al. CHK1 targets spleen tyrosine kinase (L) for proteolysis in hepatocellular carcinoma . J. Clin. Invest . 122 , 2165 - 2175 ( 2012 ).
23. Zhu , W. et al. FKBP3 promotes proliferation of non-small cell lung cancer cells through regulating Sp1/HDAC2/p27 . Theranostics 7, 3078 - 3089 ( 2017 ).
24. Zhi , X. et al. E3 ubiquitin ligase RNF126 promotes cancer cell proliferation by targeting the tumor suppressor p21 for ubiquitin-mediated degradation . Cancer Res . 73 , 385 - 394 ( 2013 ).
25. Bele , A. et al. The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells . Cell Cycle 14 , 990 - 1000 ( 2015 ).
26. Mandler , M. D. , Ku , L. & Feng , Y. A cytoplasmic quaking I isoform regulates the hnRNP F/H-dependent alternative splicing pathway in myelinating glia . Nucleic Acids Res . 42 , 7319 - 7329 ( 2014 ).
27. Jin , X. et al. An atypical E3 ligase zinc finger protein 91 stabilizes and activates NF-kappaB-inducing kinase via Lys63-linked ubiquitination . J. Biol. Chem . 285 , 30539 - 30547 ( 2010 ).
28. Wang , J. X. , Li , Q. & Li , P. F. Apoptosis repressor with caspase recruitment domain contributes to chemotherapy resistance by abolishing mitochondrial fission mediated by dynamin-related protein-1 . Cancer Res . 69 , 492 - 500 ( 2009 ).
29. Huang , J. Z. et al. A peptide encoded by a putative lncRNA HOXB-AS3 suppresses colon cancer growth . Mol. Cell . 68 , 171 - 184 e176 ( 2017 ).
30. Huang , J. Z. et al. Down-regulation of TRPS1 stimulates epithelialmesenchymal transition and metastasis through repression of FOXA1 . J. Pathol . 239 , 186 - 196 ( 2016 ).
31. Yan , G. R. et al. Genistein-induced mitotic arrest of gastric cancer cells by downregulating KIF20A, a proteomics study . Proteomics 12 , 2391 - 2399 ( 2012 ).