Oncogene

https://doi.org/10.1038/s41388-020-01527-1

ARTICLE

E3 ubiquitin ligase UBR5 promotes pancreatic cancer growth and

aerobic glycolysis by downregulating FBP1 via destabilization of
C/EBPα
Leifeng Chen1,2 Rongfa Yuan2 Chongyu Wen2 Tiande Liu2 Qian Feng2 Xueqiang Deng2 Yunyan Du
● ● ● ● ● ●
3 ●

Xiaogang Peng 4

Received: 18 July 2019 / Revised: 24 September 2020 / Accepted: 15 October 2020

© The Author(s), under exclusive licence to Springer Nature Limited 2020

Abstract
Pancreatic cancer is one of the most fatal cancers in humans. While it thrives in a state of malnutrition, the mechanism by
which pancreatic cancer cells adapt to metabolic stress through metabolic reprogramming remains unclear. Here, we showed
that UBR5, an E3 ubiquitin ligase, was significantly upregulated in pancreatic cancer patient samples compared to the levels
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in adjacent normal tissues. Levels of UBR5 were closely related to a malignant phenotype and shorter survival among
pancreatic cancer patients. Multivariate analyses also revealed that UBR5 overexpression was an independent predictor of
poor outcomes among patients with pancreatic cancer. Functional assays revealed that UBR5 contributes to the growth of
pancreatic cancer cells by inducing aerobic glycolysis. Furthermore, we demonstrated that UBR5 knockdown increased
levels of fructose-1,6-bisphosphatase (FBP1), an important negative regulator in the process of aerobic glycolysis in many
cancers. We found a significant negative correlation between levels of UBR5 and FBP1, further demonstrating that UBR5-
induced aerobic glycolysis is dependent on FBP1 in pancreatic cancer cells. Mechanistically, UBR5 regulates FBP1
expression by modulating C/EBPα, directly binding to C/EBPα, and promoting its ubiquitination and degradation. Together,
these results identify a mechanism used by pancreatic cancer cells to survive the nutrient-poor tumour microenvironment and
also provide insight regarding the role of UBR5 in pancreatic cancer cell adaptation to metabolic stresses.

Introduction

Pancreatic cancer is an aggressive malignant disease with poor

prognosis, and its death rate is almost equal to its incidence
These authors contributed equally: Leifeng Chen, Rongfa Yuan [1]. Despite significant progress, the 5-year overall survival
Supplementary information The online version of this article (https:// rate of the disease remains at about 6% [2]. Therefore, there is
doi.org/10.1038/s41388-020-01527-1) contains supplementary an urgent need for early detection methods and effective
material, which is available to authorised users. prevention strategies to reduce the mortality associated with
this disease. One obstacle underlying this clinical challenge is
* Yunyan Du
695728274@qq.com our limited understanding of how pancreatic cancer repro-
* Xiaogang Peng grams metabolism in the unique tumour microenvironment
pxg8205@163.com [3]. Thus, insights into the molecular mechanisms underlying
pancreatic cancer pathogenesis may reveal novel therapeutic
1
Jiangxi Provincial Clinical Research Center for General Surgery options for improving patient prognosis.
Disease, Nanchang, China UBR5 is an E3 ubiquitin ligase that has been implicated
2
Department of General Surgery, Second Affiliated Hospital of in the regulation of metabolism, proliferation, and apoptosis
Nanchang University, Nanchang, China [4]. Recently, emerging evidence has identified UBR5 as a
3
Department of Medical, Jiangxi Provincial People’s Hospital of key regulator of the ubiquitin–proteasome system (UPS) in
Nanchang University, Nanchang, China cancer [5–7]. Liao et al. have demonstrated that UBR5
4
Jiangxi Province Key Laboratory of Molecular Medicine, Second drives the growth and metastasis of triple-negative breast
Affiliated Hospital of Nanchang University, Nanchang, China cancer [6]. Owing to its E3 ubiquitin ligase activity, UBR5

contributes to the ubiquitination of target proteins and tags
them for degradation in many cancers [8–10]. For instance,
UBR5 promotes the progression of colorectal cancer by
destabilising the tumour suppressor ECRG4 [10]. However,
the precise role and underlying signalling cascade of UBR5
in pancreatic cancer progression remain unclear.
Aerobic glycolysis, also known as the ʻWarburg effect’, is a
hallmark of cancer characterised by the increased conversion
of glucose into lactate, irrelevant of oxygen availability [11].
Accumulating evidence indicates that aerobic glycolysis plays
an important role in the growth of various cancers, including
pancreatic cancer [12]. The regulation of glycolysis-related
proteins, such as fructose-1,6-bisphosphatase (FBP1), which
encodes a rate-limiting gluconeogenic enzyme, has been
reported to function in tumour suppression in many cancers
[13]. Loss of FBP1 expression is associated with poor prog-
nosis in patients with pancreatic cancer and hepatocellular
carcinoma [14, 15]. In particular, a prior study indicated that
inhibition of FBP1 in pancreatic cancer contributed to tumour
progression by altering glucose metabolism [14]. Interestingly,
we used RNA-seq analysis to determine that the knockdown
of UBR5 could increase FBP1 expression in PC cells (Sup-
plementary Table S1). However, the underlying mechanism
by which FBP1 is regulated in pancreatic cancer is largely
unknown. Therefore, we speculated that UBR5 could affect
the aerobic glycolysis of PC by modulating FBP1 expression.
In addition, previous studies have shown that human FBP1
is regulated by important transcription factors [16], such as
CCAAT-enhancer binding protein-α (C/EBPα) [17]. C/EBPα
is the founding member of C/EBP family, which regulates the
transcription of genes whose products are involved in pro-
gramming the gluconeogenic pathway [18]. Meanwhile, C/
EBPα is targeted for ubiquitin–proteasome-mediated degra-
dation in many cells [19]. As UBR5 is an E3 ubiquitin ligase,
it functions similarly to polyubiquitin as a tag for proteasome
targeting, but whether UBR5 is involved in regulating the
degradation of C/EBPα is largely unknown.
In the present study, we aimed to elucidate the role of
UBR5 in contributing to the prognosis of pancreatic cancer
patients. We also investigated the mechanism of this effect
with regard to FBP1 and C/EBPα in pancreatic cancer.
Taken together, our data may provide new potential prog-
nostic and therapeutic targets for pancreatic cancer.
Results
UBR5 expression is upregulated and correlated with
survival in pancreatic cancer
To investigate the role of UBR5 in the development of
pancreatic cancer, we initially explored the expression
patterns of UBR5 in pancreatic cancer tissue samples and
L. Chen et al.
adjacent non-tumour tissues. We found that the mRNA
expression level of UBR5 was significantly elevated in
tumours compared with levels in matched adjacent normal
tissues (Fig. 1A). Higher protein expression of UBR5 was
also consistently found in pancreatic cancer tissues, as
indicated by western blotting (Fig. 1B). Upregulation of
UBR5 expression in pancreatic cancer tissues was further
confirmed by IHC. As shown in Fig. 1C, D, UBR5 was
overexpressed in 66.04% (70/106) of pancreatic cancer
specimens tested, while weakly positive staining was
observed in the corresponding adjacent non-tumour tissues.
We next analysed the relationship between UBR5
expression and clinicopathological parameters in pancreatic
cancer patients. As shown in Supplementary Table S2,
UBR5 overexpression was closely correlated with TNM
stage, tumour size and histologic grade (P < 0.01) but was
not significantly correlated with age, sex, distant metastasis,
or tumour location. Furthermore, Kaplan–Meier survival
curves (Fig. 1E, F) revealed that patients with high levels of
UBR5 expression exhibited shorter overall and disease-free
survival than those with lower levels of UBR5. Multivariate
Cox regression analysis further revealed that high UBR5
expression was an independent predictor of poor survival in
patients with pancreatic cancer (Supplementary Table S3).
Taken together, these data suggest that the expression of
UBR5 is significantly upregulated in pancreatic cancer tis-
sues and implicated in the progression of pancreatic cancer.
UBR5 promotes the growth of pancreatic cancer
cells in vitro and in vivo
To further explore the function of UBR5 in pancreatic
cancer, we knocked down expression of UBR5 by stably
expressing UBR5 shRNA in PANC-1 cells, which normally
show relatively high expression of UBR5 (Fig. 2A–C).
Meanwhile, we also generated stable clones overexpressing
UBR5 from BxPC-3 cells, which exhibit relatively low
expression of UBR5 among pancreatic cancer cell lines
(Fig. 2A, B, Supplementary Fig. 1). As shown in Fig. 2E,
the results of CCK-8 assays indicated that silencing of
UBR5 clearly suppressed the growth of pancreatic cancer
cells in vitro. In contrast, UBR5-overexpressing cells
showed a significantly higher in vitro proliferation rate than
control cells (Fig. 2E). Consistent with these findings, EdU
assays showed that UBR5 knockdown decreased the cell
proliferation of PANC-1 cells, whereas overexpression of
UBR5 increased the cell proliferation of BxPC-3 cells (Fig.
2F, G). Meanwhile, we also found that UBR5 silence
reduced the cell proliferation of Rossi cells, whereas over-
expression of UBR5 increased the cell proliferation of
SW979 cells (Supplementary Fig. 2A, B).
We then subjected the UBR5-knockdown or -over-
expression cells to tumour xenograft experiments. After
E3 ubiquitin ligase UBR5 promotes pancreatic cancer growth and aerobic glycolysis by downregulating. . .
Fig. 1 Relative UBR5 expression in pancreatic cancer and its
clinical significance. A Relative mRNA levels of UBR5 in 106 pairs
of pancreatic cancer tissues and corresponding non-tumour tissues,
assessed by qRT-PCR analysis. *p < 0.05. B Determination and
quantification of UBR5 protein levels in pancreatic cancer tissues and
paired non-tumour tissues by western blotting assay. Tubulin was used
5 weeks of growth, UBR5-knockdown PANC-1 cells
(PANC-1/shUBR5) exhibited reduced tumour growth in
nude mice, whereas UBR5-overexpressing BxPC-3 cells
(BxPC-3/p-UBR5) resulted in significantly greater tumour
growth, when compared to their respective controls (Fig.
2H, I). Consistent with this, the average tumour weight in
mice bearing PANC-1/shUBR5 cells significantly
decreased, whereas that in mice bearing BxPC-3/p-UBR5
cells obviously increased, when compared to those of the
as a loading control. C, D Representative images (C) and quantifica-
tion (D) of UBR5 staining in 106 paired pancreatic cancer and non-
cancer tissues. *p < 0.05. E, F Kaplan–Meier curves for overall sur-
vival (E) and disease-free survival (F) of two groups of pancreatic
cancer patients defined by low and high expression of UBR5.
respective controls (Fig. 2J, K). In conclusion, these data
collectively indicate that UBR5 contributes to the in vitro
and in vivo growth of pancreatic cancer cells.
UBR5 induces aerobic glycolysis in pancreatic cancer
cells
Given that aerobic glycolysis is a well-characterised meta-
bolic shift that ubiquitously occurs in tumour cells including

Fig. 2 Effects of UBR5 on pancreatic cancer cell growth. A, B
mRNA and protein levels of UBR5 in pancreatic cells and the
immortalised H6c7 line. C, D the protein (C) and Mrna (D) levels of
UBR5 in PANC-1 cells after transfection with shUBR5 or shNC
(control). E CCK-8 assay showing proliferation of pancreatic cancer
cells following overexpression (left) or knockdown (right) of UBR5.
*p < 0.05. F, G Representative images (left) and quantification (right)
pancreatic cancer cells, we next explored the role of UBR5
in pancreatic cancer cell glucose metabolism. As shown in
Fig. 3A, B, knockdown of UBR5 dramatically decreased
cellular levels of glucose-6-phosphate (G6P), glucose con-
sumption, lactate production, and ATP in PANC-1 cells,
whereas UBR5 overexpression led to the opposite trends in
BxPC-3 cells.
To further validate the impact of UBR5 on pancreatic
cancer glycolysis, the extracellular acidification rate
L. Chen et al.
of EDU assays of pancreatic cancer cells transfected with p-UBR5 or
shUBR5. Scale bar, 50 μm. *p < 0.05. H, I PANC-1/shUBR5 or
BxPC-3/p-UBR5 cells were subcutaneously injected into nude mice,
and tumour volumes were measured on the indicated days. J, K At the
experimental endpoint, tumours were dissected, photographed, and
weighed (n = 6, *p < 0.05).
(ECAR), which reflects overall glycolytic flux, was mea-
sured. As shown in Fig. 3C, E knockdown of
UBR5 significantly decreased the glycolytic rate and
capacity of PANC-1 cells, whereas overexpression of
UBR5 increased ECAR in BxPC-3 cells. In addition, we
also examined OCR (cellular oxygen consumption rate), an
indicator of mitochondrial respiration. The results showed
that PANC-1/shUBR5 cells displayed an increased oxygen
consumption rate (OCR), whereas overexpression of UBR5
E3 ubiquitin ligase UBR5 promotes pancreatic cancer growth and aerobic glycolysis by downregulating. . .
Fig. 3 UBR5 promotes aerobic glycolysis in pancreatic cancer
cells. A, B Cellular G6P levels, glucose consumption, lactate pro-
duction, and ATP levels in PANC-1/shUBR5 cells (A) or BxPC-3/p-
UBR5 cells (B). Three independent experiments were performed. *p <
0.05 vs. control. C, E ECAR data showing the glycolytic rate and
capacity in UBR5-silenced (C) or -overexpressing (E) pancreatic
cancer cells. Glucose (10 mM), the oxidative phosphorylation inhibitor
oligomycin (1.0 μM), and the glycolytic inhibitor 2-deoxyglucose (2-
DG, 50 mM) were sequentially injected into each well at the indicated
time points. All measurements were normalised to the cell number
decreased the OCR in BxPC-3 cells (Fig. 3D, F). Moreover,
these responses were also exhibited in Rossi/shUBR5 and
SW979/p-UBR5 cells (Supplementary Fig. 2C, D). Taken
together, these findings demonstrate that UBR5 may pro-
mote aerobic glycolysis and inhibit mitochondrial respira-
tion in pancreatic cancer cells.
calculated using crystal violet assay at the end of the experiment. *p <
0.05 vs. control. D, F OCR results showing the basal respiration and
maximum respiration in PANC-1/shUBR5 cells (D) or BxPC-3/p-
UBR5 cells (F). Oligomycin (1.0 μM), the mitochondrial uncoupler
carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP,
1.0 μM), and the mitochondrial complex I inhibitor rotenone plus the
mitochondrial complex III inhibitor antimycin A (Rote/AA, 0.5 μM)
were sequentially injected. All measurements were normalised to the
cell number calculated using crystal violet assay at the end of the
experiment. *p < 0.05 vs. control.
UBR5 negatively regulates FBP1 protein levels
Previous studies have demonstrated that FBP1 is a negative
regulator of pancreatic cancer cell proliferation and is
related to abnormal cellular metabolism. And, the RNA-seq
data indicate that FBP1 is upregulated in UBR5-knockdown

Fig. 4 Stable knockdown of UBR5 increased FBP1 expression in
pancreatic cancer cells. A, B Western blotting and qRT-PCR ana-
lyses of FBP1 expression levels in PANC-1 cells stably transfected
with shNC or shUBR5 plasmid. *p < 0.05. C, D Western blotting and
qRT-PCR analyses of FBP1 expression levels in BxPC-3 cells stably
transfected with control vector or p-UBR5 plasmid. *p < 0.05. E, F
Determination (E) and quantification (F) of UBR5 protein levels in
cells (Supplementary Table S1). To further determine
whether UBR5 could regulate FBP1 expression, we initially
observed the protein and mRNA levels of FBP1 in UBR5-
knockdown or -overexpressing pancreatic cancer cells. As
shown in Fig. 4A, B, western blotting and qRT-PCR results
showed that UBR5 knockdown significantly increased the
protein and mRNA levels of FBP1 in PANC-1 cells. Con-
versely, overexpression of UBR5 markedly reduced the
protein and mRNA levels of FBP1 in BxPC-3 cells
(Fig. 4C, D). Next, we measured the protein levels of FBP1
in 30 pancreatic cancer tissues that had upregulated levels of
UBR5 using western blotting. Our results indicated that
protein levels of FBP1 were significantly downregulated in
pancreatic cancer tissues compared with levels in the cor-
responding adjacent normal tissues (Fig. 4E, F). Scatter
plots revealed that UBR5 and FBP1 protein expression
levels were negatively correlated in pancreatic cancer tis-
sues (Fig. 4G). In addition, qRT-PCR results also showed
L. Chen et al.
pancreatic cancer tissues (n = 30) and paired non-tumour tissues (n =
30) by western blotting. Tubulin was used as a loading control.
G Scatter plots of UBR5 and FBP1 protein expression in pancreatic
cancer. H Determination of UBR5 mRNA levels in pancreatic cancer
tissues (n = 30) and paired non-tumour tissues (n = 30) by qRT-PCR.
I Scatter plots of UBR5 and FBP1 mRNA expression in pancreatic
cancer.
that the mRNA levels of FBP1 were significantly down-
regulated in pancreatic cancer tissues compared with levels
in corresponding adjacent normal tissues (Fig. 4H), con-
sistent with the western blotting results. Furthermore, the
scatter plots showed that the UBR5 and FBP1 mRNA
expression levels were negatively correlated in pancreatic
cancer tissues (Fig. 4I). Taken together, these findings
suggest that UBR5 can reduce the expression of FBP1 and
thus promote aerobic glycolysis in pancreatic cancer cells.
FBP1 mediates UBR5-induced aerobic glycolysis in
pancreatic cancer cells
As discussed above, FBP1 was determined to be a down-
stream gene of UBR5. Next, we asked whether FBP1 is a
mediator of UBR5-induced aerobic glycolysis. Initially, we
silenced FBP1 expression in UBR5-knockdown PANC-
1 cells, and the silencing efficacy was confirmed (Fig. 5A).
E3 ubiquitin ligase UBR5 promotes pancreatic cancer growth and aerobic glycolysis by downregulating. . .
Fig. 5 Tumour-suppressive effects of UBR5 silencing in pancreatic
cancer cells partially reversed by FBP1 knockdown. A Western
blotting of UBR5 or FBP1 in PANC-1 cells stably transfected with
shUBR5 in the presence or absence of shFBP1. B CCK-8 assays
showing proliferation capacity of PANC-1 cells stably transfected with
shUBR5 in the presence or absence of shFBP1. *p < 0.05. C Quanti-
fication of EdU assays of PANC-1 cells stably transfected with
CCK-8 and EdU assays showed that the reduced pro-
liferation induced by UBR5 knockdown in PANC-1
cells was partially abolished by the introduction of
shFBP1 (Fig. 5B, C).
Next, we found that silencing FBP1 expression rescued
the UBR5-mediated reduction in cellular G6P, glucose
consumption, lactate production, and ATP levels in PANC-
1 cells (Fig. 5D). Furthermore, UBR5 knockdown
decreased ECAR in PANC-1 cells, whereas simultaneous
silencing of FBP1 attenuated the decrease in glycolytic rate
and capacity (Fig. 5E). Moreover, we measured the phy-
siological roles of FBP1 in UBR5-mediated mitochondrial
respiration. We observed that FBP1 knockdown in UBR5-
silenced PANC-1 cells counteracted the increase in OCR
induced by UBR5 knockdown (Fig. 5F). Taken together,
these findings suggest that FBP1 was involved in mediation
UBR5-induced aerobic glycolysis in pancreatic cancer cells.
shUBR5 in the presence or absence of shFBP1. *p < 0.05. D Cellular
G6P levels, glucose consumption, lactate production, and ATP levels
in PANC-1 cells stably transfected with shUBR5 in the presence or
absence of shFBP1. *p < 0.05. E ECAR of UBR5-silenced PANC-
1 cells with and without FBP1 knockdown. *p < 0.05. F OCR values
of UBR5-silenced PANC-1 cells with and without FBP1 knockdown.
*p < 0.05.
UBR5 regulates FBP1 expression by inactivating
C/EBPα in pancreatic cancer cells
UBR5 has been reported to interact with different substrates
to exert its effects. To further clarify the mechanism through
which UBR5 regulates FBP1 in pancreatic cancer cells, we
first observed whether there was a direct interaction
between the UBR5 and FBP1 proteins. However, Co-
immunoprecipitation (co-IP) showed that there was no
direct interaction between these proteins (Fig. 6A). It has
been reported that C/EBPα regulates transcription of FBP1
in hepatocellular carcinoma by binding to the two over-
lapping C/EBPα sites located at nucleotides −228/−208.
ChIP-qPCR assay also demonstrated that C/EBPα regulates
transcription of FBP1 in pancreatic cancer cells (Supple-
mentary Fig. 3). Therefore, we speculated that UBR5 reg-
ulates FBP1 via C/EBPα. To test this hypothesis, we first

Fig. 6 UBR5 regulates FBP1 expression through C/EBPα. A Co-IP
showing that endogenous UBR5 and FBP1 were not directly bound.
B Co-IP showing direct binding of endogenous UBR5 and C/EBPα in
pancreatic cancer cells. C Co-localisation studies of pancreatic cancer
cells using anti-UBR5 antibody (1:200, green) and anti-C/EBPα
antibody (1:200, red), followed by DAPI nuclear counterstaining
(blue). The merged images of UBR5 (green) and C/EBPα (red) with
DAPI (blue) are also shown. Scale bar, 50 μm. D Docking con-
formation of the first ranking score. Three-dimensional structure of
UBR5 and C/EBPα in ribbon (D1) and surface (D2) format.CEBPA is
shown in yellow. UBR5 is shown in cyan. E Protein and mRNA levels
observed whether UBR5 and C/EBPα directly interact in
pancreatic cancer cells. Interestingly, co-IP demonstrated an
interaction between UBR5 and C/EBPα (Fig. 6B). Confocal
L. Chen et al.
of FBP1 assessed by western blotting and qRT-PCR in pancreatic
cancer cells transfected with shC/EBPα or shNC. F Protein and mRNA
levels of FBP1 assessed by western blotting and qRT-PCR in pan-
creatic cancer cells transfected with p-C/EBPα or control vector.
G Protein levels of C/EBPα and FBP1 assessed by western blotting in
pancreatic cancer cells transfected with shC/EBPα or shNC. H, I
CCK-8 and EdU assays of the proliferation capacity of PANC-1 cells
stably transfected with shUBR5 in the presence or absence of shC/
EBPα. J, K Cellular G6P levels, glucose consumption, lactate pro-
duction, and ATP levels in PANC-1 cells stably transfected with
shUBR5 in the presence or absence of shC/EBPα. *p < 0.05.
microscopy was used to further confirm the co-localisation
of UBR5/C/EBPα in pancreatic cancer cells, providing
further evidence of an interaction between these two
E3 ubiquitin ligase UBR5 promotes pancreatic cancer growth and aerobic glycolysis by downregulating. . .
proteins (Fig. 6C). Furthermore, the docking results show-
ing the binding interactions between UBR5 and C/EBPα
(new Fig. 6D). Moreover, we found that purified GST-
UBR5 could bind to the HA-tagged C/EBPα under cell-free
conditions (Supplementary Fig. 4). These findings indicate
that UBR5 directly bind with C/EBPα in PC cells.
Next, to explore whether C/EBPα regulates FBP1 in
pancreatic cancer cells, we measured the changes in FBP1
expression in C/EBPα-knockdown PANC-1 cells. The
results showed that the knockdown of C/EBPα significantly
decreased FBP1 protein and mRNA expression in PANC-1
cells (Fig. 6E), whereas C/EBPα upregulation had the
opposite effect in pancreatic cancer cells (Supplementary
Fig. 5). These findings demonstrate that C/EBPα regulates
FBP1 expression in pancreatic cancer cells.
We further aimed to elucidate whether UBR5 affects the
expression of FBP1 via C/EBPα. To do this, we measured
the changes in C/EBPα and FBP1 expression in UBR5-
knockdown PANC-1 cells. The results showed that the
knockdown of UBR5 significantly increased C/EBPα and
FBP1 protein expression in PANC-1 cells (Fig. 6F) In
contrast, overexpression of UBR5 significantly decreased
C/EBPα and FBP1 protein expression in pancreatic cancer
cells (Fig. 6G). However, the mRNA level of CEBPA was
not affected changes in UBR5 expression in pancreatic
cancer cells (Fig. 6F, G). These experiments show that C/
EBPα is involved in the UBR5-mediated regulation of
FBP1 expression.
To further verify that UBR5 regulates FBP1 expression
through C/EBPα in pancreatic cancer cells, we knocked
down C/EBPα in UBR5-downregulated pancreatic cancer
cells. The results showed that the downregulation of C/
EBPα inhibited the increases in FBP1 expression, cell
proliferation, and aerobic glycolysis observed in UBR5-
knockdown PANC-1 cells (Fig. 6H–K). These results
demonstrate that the UBR5-mediated regulation of FBP1-
induced pancreatic cancer cell proliferation and aerobic
glycolysis is dependent on C/EBPα.
UBR5 destabilises C/EBPα by regulating the
ubiquitination of C/EBPα in pancreatic cancer cells
These experiments verified that UBR5 could directly bind
to C/EBPα and regulate FBP1 expression via C/EBPα. We
then further assessed the mechanisms through which UBR5
regulates C/EBPα. A study has reported that C/EBPα
undergoes degradation via the UPS. Consistently, treatment
with the proteasome inhibitor MG132 led to significant
accumulation of endogenous C/EBPα protein in pancreatic
cancer cells (Fig. 7A). Furthermore, Co-localisation results
showed that endogenous C/EBPα directly bound to ubi-
quitin in pancreatic cancer cells (Fig. 7B). Moreover, the
data indicate that efficient ubiquitination of his-C/EBPα
were detected in the presence of E1, E2 (UBCH5c), E6AP
(an E3 ubiquitin ligase for C/EBPα) and Ub (Fig. 7C). This
result demonstrates that C/EBPα is also degraded by the
UPS in pancreatic cancer cells.
Next, we studied whether UBR5 could directly mediate
C/EBPα ubiquitination. Interestingly, ectopic expression of
UBR5 led to substantially increased C/EBPα poly-ubiqui-
tination, whereas knockdown of UBR5 caused decreased C/
EBPα poly-ubiquitination (Fig. 7D, E). And, the data
showed that mutations in all Lys site of C/EBPαabolished
the C/EBPα poly-ubiquitination (Fig. 7F). As expected,
mutation of the Lys48 site on ubiquitin (Ub) almost com-
pletely abolished UBR5-mediated C/EBPα ubiquitination,
whereas the K63R mutation on Ub had no effect (Fig. 7G).
Consistent with the ubiquitination results, a degradation
dynamics assay showed that the half-life of exogenously
expressed C/EBPα was significantly increased in UBR5-
overexpressing pancreatic cancer cells compared with
that in control cells (Fig. 7H, I). These data show that UBR5
mediates Lys48-linked poly-ubiquitination of C/EBPα,
which leads to the degradation of C/EBPα in the
proteasome.
Discussion
Although advances have been made in the prognosis and
treatment of pancreatic cancer, no significant progress has
been made in improving the survival rate; it is therefore
critical to better understand the biological characteristics of
pancreatic cancer. In recent years, the importance of meta-
bolic reprogramming in the processes of tumorigenesis and
cancer development has been confirmed. Metabolic repro-
gramming is considered one of the signs of cancer [20].
Pancreatic cancer is a devastating disease with extremely
poor survival rates due to the fact that pancreatic tumours
exhibit a differential dependency on metabolism [21].
Therefore, understanding the basis of pancreatic cancer
from a metabolic perspective will provide new insights into
this disease. Here, our research demonstrated that high
expression of UBR5 predicts poor pancreatic cancer prog-
nosis and that UBR5 plays an important role in the glucose
metabolism reprogramming of pancreatic cancer.
UBR5, as a key regulator of the unfolded protein
response in development and cancer, was initially identified
in a screen for progesterone regulatory genes in human
breast cancer cells [22]. UBR5 overexpression promotes
cell proliferation in several cancers, such as gastric, cervi-
cal, and breast cancer [9, 23, 24]. A number of studies have
implicated UBR5 in various aspects of cancer biology, and
many molecular functions of UBR5 are consistent with a
role in cancer. However, no information is currently avail-
able on the specific role or molecular mechanism of UBR5

Fig. 7 UBR5 destabilises C/EBPα by promoting C/EBPα ubiqui-
tination and degradation in pancreatic cancer cells. A Pancreatic
cancer cells were treated with MG132 (Z-Leu-Leu-Leu-CHO,
15 μmol/L) for the indicated times, and levels of C/EBPα were
determined. B Co-localisation of C/EBPα and Ub in pancreatic cancer
cells. Scale bar, 50 μm. C the ubiquitination level of His-C/EBPα were
detected in the presence of E1, E2 (UBCH5c), E6AP and Ub in
HEK293 cells. D, E Knockdown or exogenous expression of UBR5
altered the ubiquitination of C/EBPα. The cells in each group were
treated with proteasomal inhibitor MG132. Cell lysates were prepared
in pancreatic cancer. In the present study, our data indicated
that the expression level of UBR5 was elevated in tumours
obtained from patients with pancreatic cancer, compared
with that in corresponding non-tumour tissues. Importantly,
high UBR5 levels were closely related to tumour size, TNM
stage, and shorter overall survival in pancreatic cancer
patients. Further investigation revealed that UBR5 con-
tributed to the growth of pancreatic cancer in vitro and
in vivo. Therefore, these findings suggest that UBR5 may
represent a novel indicator of poor prognosis in pancreatic
L. Chen et al.
and subjected to immunoprecipitation with anti-C/EBPα antibody. The
level of ubiquitin-attached C/EBPα was detected by western blotting
with anti-Ub antibody. F Ubiquitination of wild-type C/EBPα or the
K-to-R mutant (mutations in all Lys site of C/EBPα gene) in HEK293
cells. G Measurement of C/EBPα ubiquitination type in HEK293 cells.
H, I Pancreatic cells were transfected with plasmid encoding HA–C/
EBPα either with or without the Flag-UBR5 plasmid. Then, the cells
were subjected to cycloheximide (CHX) (20 μmol/L) exposure at the
indicated times, and the degradation of C/EBPα was detected with
anti-HA antibody.
cancer and may function as an oncogene in pancreatic
cancer progression.
The metabolic alterations and reprogramming in tumour
cells contribute to their survival and growth in harsh
microenvironments [25]. Thus, a better understanding of the
underlying regulatory mechanisms of aerobic glycolysis in
pancreatic cancer might aid in the discovery of novel
treatment opportunities for pancreatic cancer patients.
Recent studies have highlighted the importance of glucose
metabolism reprogramming on the growth of pancreatic
E3 ubiquitin ligase UBR5 promotes pancreatic cancer growth and aerobic glycolysis by downregulating. . .
cancer cells. For instance, miR-135 suppresses glycolysis
and promotes pancreatic cancer cell adaptation to metabolic
stress by targeting phosphofructokinase-1 [26]. Here, we
confirmed that UBR5 was closely associated with glucose
metabolism in pancreatic cancer cells. Our results revealed
that UBR5 could promote aerobic glycolysis and inhibit
mitochondrial respiration in pancreatic cancer cells. These
findings are important not only for better understanding the
biological functions of UBR5 in cancer but also for asses-
sing the potential of UBR5 as a therapeutic target.
To investigate the mechanism by which UBR5 affects
aerobic glycolysis, we focused on the mitochondrial tumour
suppressor FBP1, a key glycolytic enzyme. FBP1 is uni-
versally expressed in various tissues. Recently, many stu-
dies have revealed the significance of FBP1 in cancer
initiation and progression [27]. However, the role of FBP1
expression in pancreatic cancer development and progres-
sion remained to be determined. Herein, we have revealed a
novel mechanism for the inhibition of pancreatic cancer
proliferation and aerobic glycolysis, which occurs via an
increase in FBP1 expression mediated by UBR5 silencing.
First, we found that UBR5 expression levels are high and
FBP1 expression levels low in pancreatic cancer tissues,
with a negative correlation between the expression levels of
these proteins. Furthermore, our data demonstrated that the
downregulation of UBR5 increased FBP1 expression and
inhibited pancreatic cancer proliferation and aerobic gly-
colysis. Moreover, FBP1 downregulation rescued the
decrease in cell progression induced by UBR5 knockdown,
whereas FBP1 upregulation significantly attenuated the
increase in cell progression induced by UBR5 over-
expression. Overall, these results demonstrated that UBR5
regulates FBP1 expression to influence pancreatic cancer
progression, identifying a new regulatory mechanism
of FBP1.
Next, we further investigated the molecular mechanism
by which UBR5 regulates FBP1 expression. Research has
demonstrated that C/EBPα plays a critical role in regulating
cancer progression [28, 29]. Here, we reveal a novel
mechanism by which UBR5 regulates FBP1 expression by
affecting C/EBPα expression. This conclusion is based on
the following observations. First, our results showed that
the knockdown of C/EBPα significantly decreased FBP1
mRNA and protein expression in pancreatic cancer cells.
Second, overexpression of UBR5 significantly decreased C/
EBPα and FBP1 protein expression and increased the
transcriptional activity of C/EBPα compared with those in
the control groups. Third, the knockdown of UBR5
increased FBP1 expression, whereas downregulation of C/
EBPα attenuated this increase. Taken together, these data
demonstrate that UBR5 regulates FBP1-induced pancreatic
cancer cell proliferation and aerobic glycolysis via a
mechanism that is dependent on C/EBPα
Finally, we further investigated the mechanism by which
UBR5 regulates C/EBPα. Studies have reported that C/
EBPα is a key transcription factor [30]. The
ubiquitin–proteasome-mediated degradation of C/EBPα is
the critical mechanism by which C/EBPα levels are regu-
lated in cells [31]. In line with these results, our results
suggest for the first time that UBR5 is involved in the
degradation process of C/EBPα and that it may function as
an E3 ubiquitin ligase for C/EBPα. This conclusion is based
on the following observations. First, UBR5 is directly bond
to with C/EBPα in pancreatic cancer cells. Second, the
knockdown of UBR5 significantly reduced levels of C/
EBPα ubiquitination, whereas overexpression of UBR5
promoted C/EBPα ubiquitination. Third, UBR5 can
decrease the half-life of C/EBPα.
In conclusion, we have provided the first evidence that
UBR5 is upregulated in pancreatic cancer tissues and
associated with the progression of pancreatic cancer in
patients. We also showed that UBR5 may promote the
growth and aerobic glycolysis of pancreatic cancer cells.
More importantly, UBR5-induced aerobic glycolysis is
dependent on FBP1 in pancreatic cells. Our findings also
demonstrate that UBR5 regulates FBP1 expression by
modulating C/EBPα, which is achieved by directly binding
to C/EBPα and promoting its ubiquitination and degrada-
tion (Fig. 8). On the basis of these findings, UBR5 could
serve as a candidate biomarker for the future diagnosis and
treatment of pancreatic cancer.
Materials and methods
Tissue samples
We obtained 106 pairs of primary pancreatic cancer tumour
tissues and corresponding non-cancerous tissues from
patients undergoing resection surgery at the Department of
General Surgery, the Second Affiliated Hospital of Nan-
chang University from 2010 to 2018. All specimens from
resection surgery were frozen and stored at −80 °C for
further analysis. The clinical characteristics of all patients
are summarised in Supplementary Table S2. Written
informed consent was obtained from each patient. This
study was approved by the Ethics and Research Committees
of the Second Affiliated Hospital of Nanchang University.
Cell lines and cell culture
Four human pancreatic cancer cell lines (PANC-1, Rossi,
BxPC-3, and SW-979) and a normal epithelial kidney cell
line (H6C7) were obtained from the American Type Culture
Collection (Rockville, MD, USA). Pancreatic cancer cell
lines were routinely maintained in Dulbecco’s modified

Fig. 8 Model summarizing the
role of UBR5 in pancreatic
cancer. Proposed model by
which E3 ubiquitin ligase UBR5
promotes pancreatic cancer
growth and aerobic glycolysis
by downregulating FBP1 via
destabilization of C/EBPα.
Eagle’s medium supplemented with 10% fetal bovine serum
at 37 °C in a humidified atmosphere containing 5% CO2.
H6C7 cells were cultured in keratinocyte serum-free med-
ium (Invitrogen, Carlsbad, CA, USA) supplemented with
10% fetal bovine serum (Gibco, Grand Island, NY, USA)
and antibiotics at 37 °C in 5% CO2. All cell lines were
identified by short tandem repeat analysis performed from
the cell bank.
Quantitative real-time PCR (qRT-PCR)
RNA was reverse-transcribed using the PrimeScript RT
Reagent Kit (Invitrogen). For qRT-PCR, aliquots of double-
stranded cDNA were amplified using a SYBR Green PCR
Kit (TaKaRa, Dalian, China) and an ABI PRISM 7900
Sequence Detector (Applied Biosystems, Foster City, CA,
USA). Primer sequences were as follows: UBR5 (forward:
5′-AGGGCAATACACTGACATGG-3′; reverse: 5′-GCC
AGTGCCA TTTAGGATTC-3′), CEBPA (forward: 5′-TGG
ACAAGAACAGCAACGAGTA-3′; reverse: 5′-ATTGTC
ACTGGTCAGCTCCAG-3′), and FBP1 (forward: 5′-AC
CCGCGTCTAAAGGTTTCC-3′; reverse: 5′-GGAGGA
CTGACAGACCGAC T-3′). GAPDH (forward: 5′-CCTG
CCGGTGACTAACCCTG-3′; reverse: 5′-AGTTA AAAG
CAGCCCTGGTG-3′) was used as an internal control.
L. Chen et al.
Immunohistochemistry (IHC) and
immunofluorescent (IF) assay
Pancreatic cancer and adjacent tissues were fixed, embed-
ded in paraffin, sectioned, and deparaffinized. For IHC,
sections were blocked using serum-free protein block buffer
(Dako, Glostrup, Denmark) for 30 min and then incubated
with anti-UBR5 (1:200, Abcam), anti-C/EBPα (1:200,
Abcam), or anti-FBP1 (1:200, Abcam) antibodies. The
calculation method of IHC score was performed as
described previously [32]. In briefly, samples were exam-
ined by three individual researchers to independently obtain
pathologic information using the German semiquantitative
scoring method. Each specimen was scored for the intensity
of nucleic, cytoplasmic, and membrane staining (no stain-
ing = 0, weak staining = 1, moderate staining = 2, strong
staining = 3) and for the extent of stained cells (0% = 0,
1–24% = 1, 25–49% = 2, 50–74% = 3, 75–100% = 4). The
final IHC score was the product of the intensity score
multiplied by the extent score. The IHC score ≥5 was the
criteria for survival analyses.
For IF assays, cells were cultured on coverslips to 70%
confluency. After the indicated treatments, the cells were
fixed with 4% paraformaldehyde (PFA) and incubated at
4 °C for 30 min with 0.1% TritonX-100, then stained with
E3 ubiquitin ligase UBR5 promotes pancreatic cancer growth and aerobic glycolysis by downregulating. . .
anti-UBR5 antibody (1:100, Abcam (ab4376)) and
anti-C/EBPα (D56F10) XP® Rabbit mAb (1:100, CST
(#8178)). Then cells incubated with FITC- or tetra-
methylrhodamine isothiocyanate–conjugated secondary
antibodies (Jackson ImmunoResearch Laboratories) for
1 h at 37 °C. After that, the cell nuclei were counterstained
with DAPI (Beyotime Biotechnology, China) for 15 min.
Cells were viewed using a laser-scanning confocal
microscope (Zeiss, Germany). The confocal images
recorded on a commercial LSM700 (Zeiss; ×63/1.4 oil
objective). Single plane images were acquired with a pixel
size of 90 nm and suitable settings for the respective
dye. All images were analyzed by ImageJ software
(MD, USA).
shRNA plasmids and constructs
Plasmids encoding shRNAs against UBR5, CEBPA, and
FBP1 were synthesised by Genepharma Company
(Shanghai, China). RNAi-mediated UBR5 silencing, the
target sequences as follows: UBR5 sh#1 (5′- GCAGGA
TTGTAGGTTACTTAG-3′), UBR5 sh#2 (5′-CAACTTAG
ATCTCCTGAAA-3′). In addition, the Vectors encoding
UBR5, CEBPA, and FBP1 were also obtained from Gene-
pharma Company. Then, pancreatic cancer cells were
transfected with shRNA plasmids and vectors using Lipo-
fectamine 3000 (Invitrogen) according to the manu-
facturer’s instructions.
Western blot analysis
The western blot analysis was performed as described [33].
In brief, equal amounts of protein were separated by 10%
SDS-PAGE before transfer to nitrocellulose membranes
(Millipore), which were incubated with the primary anti-
bodies anti-UBR5 (1:1000, Abcam), anti-C/EBPα (1:1000,
Abcam), anti-FBP1 (1:1000, Abcam) and anti-Tubulin
(1:1000, Santa Cruz) at 4 °C overnight. Then, they were
blotted for 1 h at room temperature with the help of an
appropriate secondary antibody. Thereafter, ECL reagents
were used to visualise bands (Pierce, USA) and signals were
measured.
RNA sequencing
Strand-specific RNA-seq libraries were prepared using the
NEBNext Ultra Directional RNA Library Prep Kit for
Illumina (E7530L, NEB, Ipswich, MA, USA) following the
manufacturer’s instructions. Library quality was evaluated
using the Qubit® 2.0 Fluorometer (Q32866; Thermo Fisher
Scientific Inc., USA) and Bioanalyzer 2100 (Agilent).
Strand-specific RNA-seq libraries were sequenced using the
Illumina HiSeq 2500 platform.
Cell growth assays
For cell proliferation assays, multiple cultures of pancreatic
cancer cells were plated in 96-well plates at a density of 5.0 ×
103 cells/well. At the indicated time points, viable cells were
assessed using Cell Counting Kit-8 according to the manu-
facturer’s instructions. For EdU assay, the cell proliferation
rate of PC cells was determined by using a Cell-Light EdU
DNA Cell Proliferation Kit (Ribobio, Guangzhou, china) and
was calculated as the ratio of the number of EdU-positive cells
to the number of total cells (1000 cells for 10 fields of view).
Cells were seeded on coverslips in a 6-well plate. Before
detection, cells were incubated with Reagent A at a dilution of
1:1000 in culture medium for 2 h. After fixing with 4% par-
aformaldehyde, cells were incubated with fluorescent dye
(Apollo 643) for 30 min. Hoechst33342 was used to coun-
terstain the nuclei. Thereafter, the coverslips were mounted on
glass slides with Anti-fade Mounting Medium (Beyotime).
Images were captured using a Leica confocal microscope
(TSC SP8) with a ×40 objective lens. The apollo staining
signal of EdU-positive cells is completely coincident with
nuclear staining signal (Hoechst33342), or the signal coin-
cident with nuclear is significantly stronger than the signal on
cytoplasm. The percentage of EdU-positive cells was deter-
mined for comparison.
Tumourigenicity assay
Pancreatic cancer cells (1 × 106 in 100 ml PBS) were injected
subcutaneously into the flanks of nude mice (male BALB/c-
nu/nu, 6–8 weeks old). Tumour formation in nude mice was
monitored, and the tumour volume was measured every
5 days. Tumours were harvested and individually weighed
after the mice were anaesthetised. The data are presented as
tumour weight (mean ± SD). The animal work was approved
by the Ethics Committee for Animal Experiments of the
Second Affiliated Hospital of Nanchang University.
Oxygen consumption rate (OCR) and extracellular
acidification rate (ECAR)
The Extracellular Flux Analyzer XF96 (Seahorse
Bioscience, Billerica, MA, USA) was used to measure
cellular glycolysis capacity and cellular mitochondrial
respiration using the XF Cell Mito stress test kit and Gly-
colysis Stress Test Kit (Seahorse Bioscience), respectively,
according to the manufacturer’s instructions.
Co-immunoprecipitation (Co-IP) and in vivo
ubiquitination assay
Co-IP assays were performed as previously described [34].
For the in vivo ubiquitination assay, pancreatic cancer cells

with UBR5 knockdown or overexpression were exposed to
MG132 treatment for 4 h before harvesting. Then, the cell
lysate was immunoprecipitated with anti-C/EBPα antibody,
and the ubiquitination level of C/EBPα was assessed with
an anti-Ub antibody.
To the in vitro ubiquitination assays, the standard reac-
tion mixture (25 μl) contained 20 mM HEPES-NaOH (pH
7.5), 50 mM NaCl, 0.02 mg/ml BSA, 1 mM DTT, 5 mM
MgCl2, 1 mM ATP, His-C/EBPα (1 pmol as a tetramer), E1
(0.85 pmol), UBCH5c (1.25 pmol), E6AP (0.8 pmol), and
Ub (174 pmol). Reaction mixtures were prepared on ice and
then incubated at 30 °C for 10 min unless otherwise indi-
cated. The reactions were terminated by the addition of
SDS-sample buffer.
To detect C/EBPα ubiquitination in HEK293 cells, cells
were transfected with HA–C/EBPα, Myc–UBR5 and Flag-
Ub constructs for 36 h and further incubated with MG132
(15 μM) for another 4 h before analysis. In addition, the C/
EBPα mutants (mutation of all lysine residues) were used.
To assess C/EBPα ubiquitination type, two.
Ub mutants (K48R and K63R) were used. Using wild-
type human ubiquitin gene (Ub) cDNA as template, lysine
(K) at corresponding positions (K48 and K63) were mutated
into arginine. These DNA fragments were synthesised by
Genepharma Company (Shanghai, China). And, the Flag-
tagged Ub-K48R and Ub-K63R plasmids were purchased
from Genepharma Company after sequencing verification.
GST pull-down assay
GST pull-down assay was performed in HEK293 cells due
to high transfection efficiency in these cells. HA–C/EBPα
plasmid was transfected into HEK293 cells using Lipo-
fectamine 2000 (Invitrogen Life Technologies) following
manufacturer’s instructions. GST-UBR5 or control GST
(Genepharma Company, Shanghai, China) was added into
the cell lysates harvested from the cells transfected with
HA-tagged C/EBPα. After being incubated with Glu-
tathione beads (Sigma-Aldrich, St. Louis, MO, USA) for
2 h, the bound proteins were subjected to western blot
analysis using HA antibodies. For endogenous immuno-
precipitation, after being incubated with immunoglobulin G
as a control, cell lysates were incubated with protein A
beads (Sigma-Aldrich). The bound proteins were used for
western blot with UBR5 antibody.
Statistical analysis
All results are shown as mean ± SD and were analysed
using GraphPad Prism 6 (GraphPad Software, La Jolla, CA,
USA) from at least three independent experiments. The
Kaplan–Meier method was used to calculate survival
curves, and the log-rank test was used to determine
L. Chen et al.
statistical significance. Differences between groups were
analysed using two-tailed Student’s t tests and analysis of
variance (ANOVA). Data were considered statistically
significant when P < 0.05.
Funding This study was supported by grants from the National Nat-
ural Science Foundation of China (nos. 81760523, 81560117,
32060166, 81960436 and 81560396), the Project of the Jiangxi Pro-
vincial Department of Science and Technology (nos.
20171BAB215024 and 20192BCB23023), the Project of the Jiangxi
Provincial Department of Education (no. GJJ160251) and the Project
of the Jiangxi Provincial Health Commission (no. 2017A264).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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