Abstract
Aggresomes are the product of misfolded protein aggregation, and the
presence of aggresomes has been correlated with poor prognosis in
cancer patients. However, the exact role of aggresomes in tumorigenesis
and cancer progression remains largely unknown. Herein, the multiomics
screening reveal that OTUD1 protein plays an important role in
retaining ovarian cancer stem cell (OCSC) properties. Mechanistically,
the elevated OTUD1 protein levels lead to the formation of OTUD1-based
cytoplasmic aggresomes, which is mediated by a short peptide located in
the intrinsically disordered OTUD1 N-terminal region. Furthermore,
OTUD1-based aggresomes recruit ASK1 via protein-protein interactions,
which in turn stabilize ASK1 in a deubiquitinase-independent manner and
activate the downstream JNK signaling pathway for OCSC maintenance.
Notably, the disruption of OTUD1-based aggresomes or treatment with
ASK1/JNK inhibitors, including ibrutinib, an FDA-approved drug that was
recently identified as an MKK7 inhibitor, effectively reduced OCSC
stemness (OSCS) of OTUD1^high ovarian cancer cells. In summary, our
work suggests that aggresome formation in tumor cells could function as
a signaling hub and that aggresome-based therapy has translational
potential for patients with OTUD1^high ovarian cancer.
Subject terms: Cancer stem cells, Cell signalling, Mechanisms of
disease, Ovarian cancer
__________________________________________________________________
The role of aggresomes in tumorigenesis and cancer progression remains
to be explored. Here, the authors perform multi-omics and reveal that
aggresome formation supports ovarian cancer stem cell properties via
OTUD1 and ASK1/JNK signalling activation.
Introduction
Epithelial ovarian cancer is the leading cause of gynecologic
malignancy-associated death. High-grade serous ovarian cancer (HGSOC),
which is the most common histotype in the clinic, is believed to arise
from the fallopian tube^[54]1. Although initial treatment approaches
such as the combination of optimal cytoreductive surgery and
platinum-based chemotherapy are usually effective, approximately 80% of
patients experience recurrence^[55]2, and treatment options for
recurrent disease remain challenging. The presence of ovarian cancer
stem cells (OCSCs) has historically been thought to play an important
role in tumorigenicity, tumor metastasis and cancer recurrence after
initial chemotherapy^[56]3–[57]8.
The elimination of cancer stem cells (CSCs) is considered an effective
strategy for cancer treatment, which prompted us to understand how the
CSC subpopulation is maintained in tumors. Similar to normal stem
cells, CSCs are capable of self-renewal and differentiation into
heterogeneous cancer cells. The balance between self-renewal and
differentiation contributes to the abundance of CSCs^[58]9. Several
surface markers, such as CD44, CD133, CD24, ALDH and EpCAM, have been
reported for the recognition or isolation of OCSCs^[59]10–[60]15.
Moreover, several transcription factors, including NANOG, OCT4 and
SOX2, are assumed to serve as intracellular markers^[61]16. Whether
there are alterations in the expression of certain genes that are
essential for the transition of OCSCs to nontumorigenic cells is still
largely unknown. Given that the ovarian CSC subpopulation can be
maintained via different signaling pathways^[62]9,[63]17, the
elucidation of the mechanism underlying their activation would provide
valuable diagnostic biomarkers and therapeutic targets.
The combination of traditional chemotherapy and OCSC-targeted therapy
might be an effective and promising anticancer treatment approach for
ovarian cancer. The targets of interest for anti-OCSC therapy are
surface and intracellular markers of OCSCs that regulate OSCS
maintenance. Activation of signaling pathways such as the WNT^[64]18,
Hedgehog^[65]19,[66]20, Notch^[67]21, JNK^[68]22,[69]23 and MAPK^[70]24
pathways has also been shown to be associated with OCSC properties, and
targeting these signaling pathways appears to be a suitable option.
Unfortunately, clinical trials testing the anti-OCSC activity of
different pathway inhibitors either as monotherapies or in combination
with other anticancer drugs have not shown satisfactory efficacy thus
far. One of the main reasons for the unpromising results is the
intolerable toxicity of the treatments.
In this work, the deubiquitinase OTUD1 is shown to be important for
ovarian cell stemness maintenance. High levels of OTUD1 are associated
with poor prognosis in ovarian cancer patients. Our study further shows
that OTUD1 clearly forms aggresome-like organelles in the cytoplasm via
interactions in its N-terminal intrinsically disordered region; these
organelles sequester ASK1 and promote its stability, in turn activating
the downstream JNK signaling pathway to sustain OSCS maintenance. These
findings reveal that aggresome formation contributes to OSCS and plays
an important role in cancer progression and recurrence.
Results
Multiomics-based screening revealed that OTUD1 is a biomarker of poor
prognosis and is associated with CSC maintenance in ovarian cancer
We conducted this study with the aim of systematically identifying the
biomarkers driving tumorigenesis and maintaining OSCS in human ovarian
cancer. To achieve that goal, we analyzed genes that show altered gene
expression at the transcriptional level during the transition from
OCSCs to differentiated cells, factors that predict poor prognosis, and
genes that are involved in the amplified genomic regions and
susceptibility loci. Therefore, an integrated multiomics approach based
on the above aspects was developed to screen for the key factors
controlling self-renewal and stemness maintenance (Fig. [71]1a). SKOV3
cells were seeded in ultralow-attachment 96-well plates in DMEM/F12
medium supplemented with 2% B27 serum replacement to allow the
formation of floating spheres. Then, these spheres were collected and
recultured in a 6-well plate in DMEM with 10% FBS to allow the cells in
the spheres to differentiate and attach to the bottom surface,
according to a previous study^[72]25. The cell states were determined
by using qPCR to confirm the high expression of stemness-associated
genes (i.e., NOTCH1, NANOG, OCT4, SOX2, CD44) in the floating spheres
but not in the differentiated cells (Supplementary Fig. [73]1a).
Subsequently, tumor floating spheres and the derived differentiated
cells were subjected to RNA-seq analysis, and differentially expressed
genes (P < 0.05 and
[MATH: log2foldchange :MATH]
> 0.5) were identified (Fig. [74]1a). An early study identified
rs11782652, rs1243180, and rs757210 as susceptibility loci for ovarian
cancer^[75]26. We assumed that neighboring genes of those loci might be
associated with OCSC traits; thus, all of the genes within 200 kb of
those loci were chosen (Supplementary Data [76]1). Moreover, based on
human sample data in The Cancer Genome Atlas (TCGA), we identified
genes with copy number amplification (frequency >4%) (Supplementary
Data [77]2). By analyzing the intersection of the three gene sets
described above, we obtained seven candidate genes (CHMP4C, OTUD1,
SNX16, PAG1, PIP4K2A, ZFAND1, IMPA1). Among these genes, only OTUD1, a
deubiquitinase, was selected because its high level strongly predicts
poor prognosis in serous ovarian cancer (Fig. [78]1b, Supplementary
Fig. [79]1b) as well as in several other cancer types (Supplementary
Fig. [80]1c). The OTUD1 protein level decreased as the tumor spheres
derived from SKOV3 or OVCAR8 cells underwent differentiation
(Fig. [81]1c and Supplementary Fig. [82]1d and [83]1e); coincidentally,
samples of high-grade serous ovarian cancer (HGSOC), which is a poorly
differentiated subtype and possesses a higher mRNA expression-based
stemness index (mRNAsi) (Supplementary Fig. [84]1f), contained higher
levels of OTUD1 than samples of low-grade serous ovarian cancer
subtypes (Fig. [85]1d). Taken together, these results strongly suggest
that OTUD1 may act as a proto-oncogene in ovarian cancer and potentiate
OSCS maintenance.
Fig. 1. OTUD1 is identified to be associated with OSCS maintenance and its
high expression positively correlate with histological high-grade ovarian
serous cancers.
[86]Fig. 1
[87]Open in a new tab
a A multiomic integration approach was developed to screen the key
factor controlling self-renewal and maintenance of OCSC. SKOV3 floating
spheres and the derived differentiated cells were subjected to RNA-seq
analysis, which is sorted by
[MATH:
−log10PValue :MATH]
. The obtained differentially expressed genes were further screened
based on the gene dataset of susceptibility loci for ovarian cancer and
their negatively correlation with tumor prognosis. SDC Sphere
Differentiated Cells. FS Floating Sphere. b Kaplan–Meier survival
curves revealed a negative correlation between OTUD1 expression level
and progression-free survival (PFS) in individuals with serous ovarian
cancer ([88]https://kmplot.com/analysis/). n = 535 patients with serous
ovarian cancer. c The protein level of OTUD1 were measured in SKOV3
floating spheres and the derived differentiated cells. SDC Sphere
Differentiated Cells. FS Floating Sphere. d Immunohistochemistry
analysis of OTUD1 protein abundance in ovarian serous cancer tissue
microarray; representative images of indicated pathological grade are
shown (Scale bar, 20 μm). LGSOC samples, n = 4; HGSOC samples, n = 54.
HGSOC, High grade serous ovarian carcinoma. LGSOC Low grade serous
ovarian carcinoma. Representative images (e) and statistical analysis
(f) demonstrating the effects of OTUD1 knockout on floating
sphere-forming capacity in SKOV3 and OVCAR8 cell lines (n = 3). Scale
bar, 50 μm. g Flow cytometry analysis of CD44^+ and CD133^+ CSCs in
SKOV3 OTUD1 depleted cell line. Numbers in the charts indicate the
percentages of corresponding subpopulations (n = 3). Tumor images (h)
and volumes (i) in mice injected with negative control and OTUD1
knockout SKOV3 cells (n = 6 mice per group). Tumor sizes were monitored
and the protein level of xenografts were analyzed by western blot (j).
P values are calculated using two-tailed unpaired Student’s t test (d),
one-way ANOVA (f, g) and two-way ANOVA (i). n.s. not significant.
Representative of n = 3 independent experiments (c, e, g). Source data
are provided as a Source Data file.
OTUD1 knockout effectively decreased tumorigenicity and OSCS but not the
proliferation rate of ovarian cancer cells
To verify our hypothesis, we knocked out OTUD1 in the ovarian cancer
cell lines SKOV3, OVCAR8 and CAOV3 by using two distinct sgRNAs
(Supplementary Fig. [89]1g). Relative to control cells, cells with
OTUD1 knockout (KO) resulted in compromised tumor sphere formation
(Fig. [90]1e, f, and Supplementary Fig. [91]1h), soft agar colony
formation (Supplementary Fig. [92]1i) and tumor cell invasion ability
(Supplementary Fig. [93]1j). In addition, a flow cytometric cell
sorting assay showed that loss of OTUD1 obviously decreased the cancer
stem cell ratio (Fig. [94]1g). Finally, a mouse xenograft tumor model
was used to determine the effects of OTUD1 KO on tumorigenesis in vivo.
The results showed that compared with controls, tumors derived from
knockout cells grew more slowly and were much smaller (Fig. [95]1h–j,
and Supplementary Fig. [96]1k-l). Interestingly, depletion of OTUD1 did
not affect SKOV3 cell growth (Supplementary Fig. [97]1m), implying that
OTUD1 probably contributes to cancer progression through its effect on
OSCS.
OTUD1 is involved in MAPK/JNK pathway regulation and physically interacts
with ASK1
To determine the mechanism by which OTUD1 promotes OSCS maintenance, we
performed RNA-seq using SKOV3 cells engineered to express sgNC or
sgOTUD1. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway enrichment analysis of the identified differentially expressed
genes, several pathways, including the MAPK, AKT, Rap1, and focal
adhesion pathways, were enriched (Fig. [98]2a). Given that the KEGG
analysis showed MAPK pathway enrichment and that many previous studies
reported the importance of MAPK/JNK activation in ovarian CSC activity
regulation^[99]27–[100]29, although it may play different roles in
different cancer types^[101]30–[102]33, we focused on the effect of
OTUD1 on the MAPK/JNK pathway. p-ERK, p-p38 and p-JNK levels in OTUD1
knockout cells were determined. The results showed that only p-JNK was
significantly downregulated (Fig. [103]2b). Indeed, OTUD1 KO
downregulated the expression of several reported JNK target genes and
CSC-associated genes, which was confirmed by qPCR (Fig. [104]2c, and
Supplementary Fig. [105]2a, b). Because OTUD1 can typically stabilize
proteins via its deubiquitinase activity, to identify the possible
proteins in the JNK pathway that are targeted by OTUD1, we first
detected the changes in the protein levels of several main JNK
components in the presence of OTUD1. An EGFP-OTUD1 inducible system was
then constructed to evaluate the effect of OTUD1 on JNK pathway
proteins. As shown in Fig. [106]2d, ectopic OTUD1 expression
upregulated apoptosis signal-regulation kinase-1 (ASK1) and increased
ASK1 phosphorylation levels. ASK1 is a mitogen-activated protein kinase
family member (MAP3K5) that activates JNK pathways in response to
intracellular and extracellular stimuli^[107]34, and high ASK1 mRNA
expression predicts poor prognosis in ovarian cancer (Supplementary
Fig. [108]2c). Moreover, JNK phosphorylation was also significantly
increased (Fig. [109]2d). To firmly establish a link between JNK and
the maintenance of stemness, we treated OTUD1-expressing cells with
T-5224 (T-5), which is a selective c-Jun inhibitor. Consistently,
T-5224 treatment significantly compromised the promoting effect of
ectopic OTUD1 on the expression of key CSC genes (Supplementary
Fig. [110]2d). In addition, loss of OTUD1 reduced the c-Jun
phosphorylation level in SKOV3, OVCAR8 and CAOV3 cells (Supplementary
Fig. [111]2e), implying that OTUD1 probably regulates OSCS by
activating the JNK/c-Jun pathway.
Fig. 2. OTUD1 plays a role in MAPK/JNK pathway regulation and physically
interacts with ASK1.
[112]Fig. 2
[113]Open in a new tab
a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the
differentially expressed genes between sgRNA-OTUD1 and control cells
(sgCtrl). The top 9 enriched pathways were listed. The bubble size
indicates changed gene numbers and colors represent false discovery
rate (P‐value). b The specific effect of OTUD1 on the phosphorylation
levels of several key components of the MAPK signaling pathway
(ERK/p-ERK, p38/p-p38, JNK/p-JNK) were determined. c The expression of
CD24, FOS and several other reported JNK target genes (IL6 and ATF2),
as well as a number of CSCs markers such as OCT4, NANOG and NOTCH1 in
SKOV3 control or OTUD1 depleted cells, were analyzed by qPCR (n = 3). d
A dox-inducible EGFP-OTUD1 expression system was established to
evaluate the effects of OTUD1 on protein levels of JNK signal pathway
key components (ASK1/p-ASK1, JNK/p-JNK). Dox doxorubicin.
e Co-immunoprecipitation (Co-IP) was performed with Flag-OTUD1 WT and
HA-ASK1 in 293T cells. f Co-immunoprecipitation (Co-IP) was performed
with Flag-OTUD1 WT or Flag-OTUD1 C320A mutant and HA-ASK1 in 293T
cells. C320A, OTUD1-C320A. g Schematic illustration of OTUD1 domain.
OTU ovarian tumor protease, UIM ubiquitin-interacting motif, IDR
intrinsically disordered protein region. h Co-immunoprecipitation
(Co-IP) was performed with Flag-OTUD1 (aa 1–286 or aa 287–481
truncation) and HA-ASK1 in 293T cells. P values are calculated using
one-way ANOVA (c). Representative of n = 3 independent experiments (b,
d–f, h). Source data are provided as a Source Data file.
Moreover, depletion of OTUD1 reduced the ASK1 phosphorylation level in
SKOV3 and OVCAR8 cells (Supplementary Fig. [114]2f). We then examined
the interaction between OTUD1 and ASK1. Reciprocal
coimmunoprecipitation (Co-IP) revealed that OTUD1 binds to ASK1
(Fig. [115]2e), and their interaction was not influenced by the
enzymatically inactive OTUD1 C320A mutant (Fig. [116]2f). To
characterize the domains involved in the protein interaction, a series
of truncated constructs were generated. As shown in Fig. [117]2g, h,
both the N-terminus and C-terminus of OTUD1 bound to ASK1. These
results prompted us to investigate whether OTUD1 is sufficient to
activate ASK1 downstream signaling and whether its deubiquitinase
activity is needed for this regulation.
OTUD1 promotes ASK1 stability in a largely deubiquitinase-independent manner
As a deubiquitinase, wild-type (WT) OTUD1 but not the C320A mutant
efficiently reduced ASK1 K48 ubiquitination (Fig. [118]3a), implying
that OTUD1 inhibits ASK1 degradation in a proteasome-dependent manner.
The EGFP-OTUD1-WT (WT) and EGFP-OTUD1-C320A (C320A) inducible plasmids
were then constructed to evaluate the effect of OTUD1 on ASK1 levels.
As shown in Fig. [119]3b, ectopic expression of WT OTUD1 increased the
ASK1 level, consistent with the observation that OTUD1 reduced ASK1 K48
ubiquitination. Surprisingly, expression of the OTUD1
deubiquitinase-dead C320A mutant increased the ASK1 protein level to an
extent comparable to that resulting from WT OTUD1 expression
(Fig. [120]3b). We therefore speculated that OTUD1 might stabilize ASK1
in an essentially deubiquitinase-independent manner.
Fig. 3. Elevated OTUD1 protein forms aggresome and re-localize ASK1 in the
cytoplasm.
[121]Fig. 3
[122]Open in a new tab
a IB analysis of input and IP products from 293T cells transfected with
Flag-OTUD1 (Flag-WT) or Flag-C320A, ubiquitin-myc (Ubi-myc) or
Ubi-K48O-myc, and HA-ASK1. The cell lysates were treated with protease
inhibitor MG132 prior to Co-IP experiment. WT, OTUD1-WT; C320A,
OTUD1-C320A. b Two dox-inducible EGFP-OTUD1-WT and EGFP-OTUD1-C320A
expression system were established to assess the effect of OTUD1 on
ASK1 protein levels. Dox, doxorubicin. c Representative
immunofluorescence images of 293T cells transiently transfected with
EGFP and EGFP-OTUD1 WT or EGFP-C320A plasmids. Scale bar, 25 μm. d
Representative images of fluorescence recovery at prebleach (0 s) and
postbleach (30 s, and 90 s). Scale bar, 10 μm. e Representative
immunofluorescence images of indicated 293T cells treated with NOCO.
Scale bar, 25 μm. NOCO, nocodazole. f Immunofluorescence was performed
to observe the co-localization of EGFP-OTUD1 and HDAC6 (aggresome
biomarker). Scale bar, 25 μm. g Representative immunofluorescence
images of 293T cells co-transfected with mcherry-ASK1 and EGFP-OTUD1 or
EGFP-C320A plasmid. Scale bar, 25 μm. h A dox-inducible 293T EGFP-OTUD1
expression system was generated. The resultant cells were transfected
with mcherry-ASK1 and treated with doxorubicin (DOX) (1 μM) for 24 h
before the media was changed. Subsequently, the aggregation of OTUD1
and ASK1 was observed by Laser Scanning Confocal Microscope at 0, 12 h,
24 h, and 48 h after DOX (1 μM) was removed. Scale bar, 5 μm. Dox
doxorubicin. Representative of n = 3 independent experiments (a–h).
Source data are provided as a Source Data file.
Elevated OTUD1 protein levels induce the formation of aggresome-like
organelles and relocalizes ASK1
We first investigated the cellular location of OTUD1 by using an
EGFP-tagged construct. Unexpectedly, when overexpressing EGFP-OTUD1-WT
or C320A, we observed an area of protein aggregation with an irregular
border in the cytoplasm in most of the cells with EGFP signals (the
percentage of the aggregates was 0% for the control, 75.0% for WT and
66.4% for C320A; the mean diameter of the aggregates was 0 μm for the
control, 9.2 μm for WT and 8.2 μm for C320A (Fig. [123]3c, and
Supplementary Fig. [124]3a). To characterize the properties of the
aggregation, we performed a fluorescence recovery after photobleaching
(FRAP) assay. As shown in Fig. [125]3d and Supplementary Fig. [126]3b,
live 293T cells transiently transfected with EGFP-OTUD1 WT were
subjected to laser microirradiation. FRAP was performed up to 90 s
after microirradiation, and no EGFP signal recovery was observed at the
irradiation site, suggesting that the aggregation was unlikely to be a
result of phase separation. Consistent with these findings, when cells
were treated with 1,6-hexanediol (1,6-HDO), a phase separation
inhibitor, aggregation was not affected (Supplementary Fig. [127]3c).
We then hypothesized that if the aggregation was an aggresome, which is
normally formed depending on the presence of microtubules^[128]35. To
verify this hypothesis, we treated cells with nocodazole (NOCO), a
microtubule inhibitor, to disrupt microtubules and block the formation
of aggresomes. The aggregation dramatically disappeared in response to
nocodazole treatment, indicating that the ectopic OTUD1-mediated
aggregates were probably aggresomes (Fig. [129]3e). To further prove
this hypothesis, we investigated the colocalization of OTUD1 and
several aggresome markers, namely, HDAC6, P62 and HSP70, and the
results showed that OTUD1-based aggresomes were colocalized with all of
these markers very prominently (Fig. [130]3f, Supplementary
Fig. [131]3d–e), reinforcing the idea that OTUD1 can form aggresomes
without additional proteasome inhibition. Because OTUD1 strongly bound
to ASK1, we sought to determine whether ASK1 could be recruited to
OTUD1-based aggresomes. As shown in Fig. [132]3g, compared with the
observations in control cells, ASK1 aggregated upon OTUD1
overexpression, and both WT OTUD1 and the C320A mutant completely
colocalized with ASK1. An EGFP-OTUD1 inducible plasmid was then
constructed to evaluate the dynamics of the colocalization of OTUD1 and
ASK1. As shown in Fig. [133]3h and Supplementary Fig. [134]3f, after
treating 293T cells with doxorubicin (DOX) for 24 h, OTUD1-based
aggresomes were observed, and they were colocalized with ASK1.
Interestingly, the aggresomes gradually disappeared upon DOX removal,
accompanied by a large decrease in the size of the ASK1 condensate.
Moreover, we treated cells expressing OTUD1 with DMSO or NOCO. As shown
in Supplementary Fig. [135]3g, NOCO treatment gradually eliminated
OTUD1 aggresome formation, accompanied by a reduction in ASK1 puncta,
suggesting that ASK1 aggregation is completely dependent on OTUD1-based
aggresome formation. Because aggresomes are partially resistant to
proteasomal degradation, we assumed that ASK1 sequestration by
OTUD1-based aggresomes significantly promotes ASK1 protein stability
and, in turn, activates the downstream JNK pathway.
The N-terminal intrinsically disordered region of OTUD1 is critical for its
supramolecular assembly, aggresome formation, and associated ASK1 recruitment
Previous studies showed that proteasome inhibition can facilitate
aggresome formation by several proteins; however, our results provide
evidence that an elevated OTUD1 protein level alone was sufficient to
induce aggresome formation, implying that OTUD1 might undergo a
self-supramolecular assembly process. We first examined the possible
oligomerization of the full-length, N-terminus, and C-terminus of the
OTUD1 protein. The results clearly showed that both full-length OTUD1
and the N-terminus but not the C-terminus could form complexes because
Flag- and HA-tagged OTUD1 interacted with each other (Fig. [136]4a, b
and Supplementary Fig. [137]4a). Given that aggresome formation is
primarily caused by the presence of misfolded proteins, we sought to
determine whether OTUD1 contains a unique disordered region that
contributes to both its supramolecular assembly and protein
aggregation. Indeed, OTUD1 contains an N-terminal intrinsically
disordered region (IDR), which was also predicted by the AlphaFold
Protein Structure Database (Fig. [138]4c). After several rounds of
screening, an OTUD1 N-terminal peptide located between amino acids (aa)
105 and 164 was shown to be responsible for OTUD1 complex formation
(Fig. [139]4d and Supplementary Fig. [140]4b–e). Furthermore, while
supramolecular assembly of the purified native WT OTUD1 protein was
observed in vitro, deletion of aa 105–164 (M1) almost completely
abolished OTUD1 aggregation (Fig. [141]4e).
Fig. 4. A peptide locates in OTUD1 N-terminal intrinsic disordered region is
critical for aggresomes formation and ASK1 recruitment.
[142]Fig. 4
[143]Open in a new tab
a, b IB analysis of input and IP products from 293T cells transfected
with Flag- and HA-tagged OTUD1. c The predicted three-dimensional
protein structure of OTUD1 was obtained from AlphaFold Protein
Structure Database ([144]https://alphafold.ebi.ac.uk/entry/Q5VV17). d
IB analysis of input and IP products from 293T cells transfected with
full-length (OTUD1-WT) or OTUD1 IDR mutant plasmid (Δ105–164, M1). IDR,
intrinsically disordered protein region. WT, OTUD1-WT; M1,
OTUD1-Δ105–164. e Gel-shift experiments showing the super-assembly of
the purified OTUD1 protein (WT) and Δ105–164 deletion mutant protein
(M1). f Dox-inducible EGFP-OTUD1 (EGFP-OTUD1^WT) and
EGFP-OTUD1-Δ105–164 (EGFP-OTUD1^M1) expression plasmids were
constructed and transfected into 293T. Representative images of
Immunofluorescence on 293T cells co-transfected with mcherry-ASK1 and
EGFP-OTUD1^WT or EGFP-OTUD1^M1 plasmid. Scale bar, 10 μm. Statistical
analysis showing the percentage of cells containing observable
aggresome and their average diameter of OTUD1 (WT) or mutant OTUD1
proteins (Δ105–164, M1). n = 7 EGFP positive cells examined across 3
independent experiments. Dox, doxorubicin. g The dox-inducible
EGFP-OTUD1 (EGFP-OTUD1^WT) and EGFP-OTUD1-Δ105–164 (EGFP-OTUD1^M1)
expression system was constructed. IB analysis showing the indicated
proteins (ASK1, p-ASK1, JNK, p-JNK, c-Jun and p-c-Jun) in SKOV3 cells
upon OTUD1^WT (WT) or OTUD1^Δ105-164 (M1) expression. Dox doxorubicin,
EV empty vector. h IB analysis of input and IP products from 293T cells
co-transfected with N-terminus of ASK1 plasmid (Flag- or HA-tagged
ASK1) and Flag-OTUD1 to detect the ASK1 dimerization. N, N-terminus. i
IB analysis of input and IP products from 293T cells transfected with
N-terminus of ASK1 plasmid (Flag- or HA-tagged ASK1), Flag-OTUD1^WT
(WT), and Flag-OTUD1^Δ105-164 (M1). N, N-terminus. P values are
calculated using two-tailed unpaired Student’s t test (g).
Representative of n = 3 independent experiments (a, b, d–i). Source
data are provided as a Source Data file.
An EGFP-OTUD1 inducible system was again used to evaluate the dynamics
of the colocalization of OTUD1 and ASK1. As shown in Fig. [145]4f,
after treating 293T cells with doxorubicin (DOX) for 12 h, deletion of
aa 105–164 disrupted the tendency of OTUD1 to form aggresomes
colocalized with ASK1. Accordingly, the expression of Δ105–164 failed
to stabilize the ASK1 protein as OTUD1^WT did, reinforcing the idea
that the recruitment of a protein to aggresomes may enhance its
resistance to proteasomal degradation. We then further explored whether
the colocalization of ASK1 and OTUD1 affects ASK1 and other JNK protein
activities. As shown in Fig. [146]4g, after treating SKOV3 cells with
doxorubicin (DOX) for 12 h, ectopic expression of OTUD1 but not
Δ105–164 (M1) increased the phosphorylation levels of ASK1 at Thr838
and the expression levels of the downstream phosphorylated forms of JNK
and c-Jun, all of which indicate ASK1 activation. Moreover, we observed
enhanced dimerization of ASK1, which is essential for ASK1 activation,
most likely because of enforced proximity (Fig. [147]4h). Consistent
with these findings, depletion of aa 105–164 in OTUD1 attenuated ASK1
dimerization (Fig. [148]4i), suggesting that ASK1 recruitment mediated
by aggresomes not only increased its protein stability but also
maintained its activity. In addition, we performed an
immunofluorescence experiment to detect whether proteins sequestered in
the aggresome could be functional or continuously activated. As shown
in Supplementary Fig. [149]4f-[150]4g, enhanced p-ASK1 and p-JNK signal
intensities were observed. Moreover, we found that both p-ASK1 and
p-JNK were enriched in OTUD1 WT-based aggresomes, while deletion of
OTUD1 105–164 aa (M1) failed to sequester p-ASK1 and p-JNK, suggesting
that those proteins sequestered in the aggresome could be functional or
continuously activated. All the above results demonstrated that
OTUD1-based aggresomes contribute to ASK1 protein stabilization and
persistent ASK1/JNK activation and that this function is mediated by a
region (aa 105–164) located in the OTUD1 N-terminal intrinsically
disordered region.
Abrogation of OTUD1 aggresome formation impaired the stimulatory effect of
OTUD1 on OSCS maintenance
Because the active JNK pathway has been shown to be involved in CSC
maintenance by numerous studies^[151]22,[152]36, we focused on the
stimulatory effect of both OTUD1^WT and OTUD1^Δ105-164 on OCSCs. To
achieve this goal, we initially performed a xenotransplantation
limiting dilution assay to investigate the effects of OTUD1^WT or
mutant on the OSCS. A total of 1 × 10^4, 1 × 10^5, or 1 × 10^6 SKOV3
cells (EV, OTUD1^WT, OTUD1^∆105-164) were counted and subcutaneously
injected into BALB/c nude female mice. The results showed that
OTUD1^WT-expressing cells exhibited much higher tumor initiation rates
and greater tumor growth potential than control cells. In contrast, the
deletion of aa 105–164 markedly impaired the induced tumorigenicity,
which was reflected by reductions in both the average tumor volume and
number (Fig. [153]5a–c). Next, the CD133^+ and CD44^+ cell proportions
in each group were determined. The results showed that ectopic
expression of OTUD1^WT but not OTUD1^∆105-164 in SKOV3 cells greatly
increased the CD133^+ and CD44^+ cell proportions (Fig. [154]5d and
Supplementary Fig. [155]5a). Similarly, expression of OTUD1^WT but not
OTUD1^∆105-164 in SKOV3 cells promoted sphere formation and
anchorage-independent growth (soft agar assay) (Fig. [156]5e, f and
Supplementary Fig. [157]5b–d). Accordingly, the expression of OTUD1^WT
but not the ∆105–164 mutant upregulated the expression of several OCSC
marker genes (Fig. [158]5g). Rescue experiments by re-expressing WT- or
M1-OTUD1 in SKOV3 OTUD1-depleted cells were performed to exclude
possible off-target effects. As shown in Supplementary Fig. [159]5e–g,
WT-OTUD1 but not the M1 mutant re-expression increased sphere formation
and colony formation. These results suggested that OTUD1-based
aggresomes are critical for maintaining OSCS. In addition, OTUD1 and
ASK1 subcellular localization was analyzed in the xenograft tumor
tissues. The results showed that OTUD1^WT can aggregate and essentially
colocalize with ASK1, while Δ105–164 caused almost no protein
aggregation (Fig. [160]5h, i). IHC analysis of xenograft tumors also
revealed that OTUD1^WT but not OTUD1^∆105–164 increased ASK1 protein
levels and JNK phosphorylation (Supplementary Fig. [161]5h). These
results strongly suggest that the ASK1/JNK pathway is a therapeutic
target in OTUD1-high OCSCs.
Fig. 5. Aggresome formation of OTUD1 is critical for promoting OSCS
maintenance.
[162]Fig. 5
[163]Open in a new tab
1 × 10^4, 1 × 10^5 or 1 × 10^6 SKOV3 cells were subcutaneously injected
into BALB/c nude female mice (n = 6 mice per group). After 25, 32, or
54 days, the mice were sacrificed and the tumor tissues were collected
as shown in a. The table shows the number of harvested tumors in each
group (b). Tumor growth curve of indicated xenografts are created based
on tumor volume of indicated time (c). EV empty vector. d Effect of WT
OTUD1 or Δ105–164 mutant on CD44^+ or CD133^+ cells proportions were
evaluated by flow cytometry analysis. EV empty vector. e Representative
images of the effects of depletion of OTUD1 IDR region (105–164 aa) on
sphere-formation in SKOV3 cells. Scale bar, 50 μm. IDR intrinsically
disordered protein region. EV empty vector. f Representative images of
the effect of WT (OTUD1^WT) or IDR depleted mutant OTUD1
(OTUD1^Δ105–164) on anchor-independent growth capacity in SKOV3 cells.
Scale bar, 400 μm. EV empty vector. g qPCR was employed to confirm the
expression of stemness-associated genes (ie, CD133, CD44, CD24, OCT4,
SOX2, NOTCH1) in SKOV3 OTUD1^WT or OTUD1^Δ105-164 expressing cells
(n = 3). EV empty vector. h Immunofluorescence analysis was performed
to examine the correlation of OTUD1^WT or OTUD1^Δ105–164 expression and
ASK1 protein level or distribution in vivo. Scale bar, 15 μm. i
Statistical analysis showing the OTUD1-based aggresome average diameter
of OTUD1 (WT) or mutant OTUD1 proteins (Δ105–164, M1) in SKOV3
xenografted tumor samples (n = 25 cells examined over 2 xenografted
tumor samples). EV, empty vector. P values are calculated using two-way
ANOVA (c), one-way ANOVA (g), two-tailed unpaired Student’s t test (h).
Representative of n = 3 independent experiments (d–f). Source data are
provided as a Source Data file.
ASK1/JNK pathway inhibitors effectively suppressed OTUD1-mediated OSCS
Targeting the JNK signaling pathway has been recognized as a strategy
in cancer therapy, and several JNK inhibitors have been developed.
However, the potency of these inhibitors is limited due to the lack of
biomarkers. IN-8 is a specific inhibitor of the Jun N-terminal kinase.
Selonsertib (SE) is a selective ASK1 inhibitor that was tested for NASH
treatment in a clinical trial^[164]37. Moreover, a recent study showed
that the burton tyrosine kinase inhibitor ibrutinib, which has been
approved by the FDA for the treatment of lymphoma and chronic
lymphocytic leukemia, is an MKK7 inhibitor that can inhibit the
ASK1/JNK pathway^[165]38. We therefore tested the effect of these
inhibitors on OSCS driven by OTUD1.
As shown in Fig. [166]6a–d, Supplementary Figs. [167]6a, b and [168]7a,
b, OTUD1 promoted sphere formation and anchorage-independent growth in
soft agar, while the promoting effect was significantly compromised by
administration of IN-8, SP600125, selonsertin or ibrutinib, as
determined by the downregulated expression of key CSC genes and reduced
CSC content (Fig. [169]6e–g, Supplementary Figs. [170]6c and [171]7c),
indicating that targeting the ASKI/JNK pathway is a potential
therapeutic strategy for OTUD1^high ovarian cancer. In addition,
platinum-based chemotherapy efficacy is determined by the presence of
CSCs. Indeed, our results showed that OTUD1 expression reduced the
sensitivity of SKOV3, OVCAR8 and CAOV3 cells to cisplatin treatment,
most likely by promoting OSCS maintenance (Fig. [172]6h, i,
Supplementary Figs. [173]6d–g and [174]7d). Interestingly, treatment
with IN8, SP600125 or ibrutinib essentially enhanced the sensitivity to
platinum-based (cisplatin, DDP) chemotherapy, suggesting that the
combination of ASK1/JNK pathway inhibition and platinum-based
chemotherapy might represent a therapeutic strategy in ovarian cancer.
These results also suggested that persistent ASK1/JNK activation is
important for OTUD1^high OSCS. Because ibrutinib is an FDA-approved
drug with tolerable toxicity, we focused on the effect of ibrutinib on
the tumorigenicity of OTUD1-high ovarian cancer cells.
Fig. 6. ASK1/JNK inhibitors potentially suppressed stemness of OTUD1^high
ovarian cancer cells.
[175]Fig. 6
[176]Open in a new tab
a Representative images demonstrate the effects of IN-8 (2 μg/mL),
selonsertib (SE, 1 μM), or ibrutinib (2.5 μg/mL) treatment on SKOV3
sphere-formation with or without ectopic OTUD1, respectively. EV empty
vector. Scale bar, 50 μm. b Statistical analysis reveals the effect of
inhibitors (IN8, Selonsertib, and Ibrutinib) on number of floating
spheres of indicated SKOV3 cells in Fig. 6a (n = 3). EV empty vector,
SE selonsertib. c The soft agar assay showing anchorage-independent
growth of OTUD1 cells in SKOV3 treated with or without IN-8 (2 μg/mL),
Selonsertib (SE, 1 μM), or Ibrutinib (2.5 μg/mL). Scale bar, 400 μm. d
Statistical analysis illustrates the effect of inhibitors (IN8,
Selonsertib, and Ibrutinib) on number of colonies in indicated SKOV3
cells in Fig. 6c (n = 3). EV empty vector, SE selonsertib. e The qPCR
assay was utilized to confirm the expression of stemness-associated
genes (i.e., CD44, OCT4, and SOX2) in SKOV3 cells expressing OTUD1 with
indicated treatment (n = 3). EV empty vector, SE selonsertib. f Flow
cytometry analysis of CD44^+ and CD133^+ CSCs in SKOV3 OTUD1^WT cell
with indicated treatment. Numbers in the charts indicate the
percentages of corresponding subpopulations. EV empty vector. g
Statistical analysis showing the effect of those three inhibitors (IN8,
Selonsertib, and Ibrutinib) on the percentages of CSCs in Fig. 6f
(n = 3). EV empty vector. h The chemotherapy sensitivity assay by using
SKOV3 cells expressing OTUD1 with or without IN-8 (2 μg/mL), Ibrutinib
(2.5 μg/mL), or/and DPP (1 μg/mL) treatment. DPP Cisplatin, EV empty
vector. i Statistical analysis displays the effect of those three
inhibitors (IN8, Selonsertib, and Ibrutinib) on the clonal formation in
Fig. 6h (n = 3). EV empty vector. P values are calculated using
unpaired Student’s t test (b, d, e, g), one-way ANOVA (i).
Representative of n = 3 independent experiments (a, c, f, h). Source
data are provided as a Source Data file.
The FDA-approved drug ibrutinib preferentially reduces the tumorigenicity of
OTUD1^high ovarian cancer cells
To further verify the effect of ibrutinib, we selected several ovarian
cancer cell lines, and SKOV3 cells were shown to contain a relatively
higher protein abundance of OTUD1 than OVCAR3 cells, accompanied by
elevated protein levels of ASK1, p-ASK1, p-JNK and p-c-Jun
(Fig. [177]7a). In addition, higher OTUD1, ASK1 and p-ASK1 levels were
also found in OVCAR8 cells than in OVCAR3 cells (Supplementary
Fig. [178]8a). Consistent with the above findings, OTUD1 aggregated and
colocalized with ASK1 in SKOV3 cells but not in OVCAR3 cells
(Fig. [179]7b). Moreover, knocking out OTUD1 in SKOV3 cells abrogated
ASK1 aggregation (Fig. [180]7c). Both SP600125, IN-8 and ibrutinib much
more efficiently inhibited sphere formation in both SKOV3 and OVCAR8
cells than in OVCAR3 cells (Fig. [181]7d, e and Supplementary
Fig. [182]8b–f). Notably, ibrutinib dramatically suppressed tumor
growth in a xenograft model established with SKOV3 cells and OVCAR8
cells; in contrast, it only modestly affected OVCAR3 cell-derived tumor
growth (Fig. [183]7f and Supplementary Fig. [184]8g). Immunoblotting
(IB) revealed that the JNK/c-Jun pathway was inhibited by ibrutinib
(Fig. [185]7g). These results strongly suggested that a high level of
OTUD1 in ovarian cancer could serve as a useful biomarker for
ibrutinib-based OCSC-targeted therapy. To further support this idea, we
analyzed the OTUD1 and ASK1 protein levels and distribution in high-
and low-grade serous ovarian cancer tissues, and the results revealed
that OTUD1 preferentially aggregated and colocalized with ASK1 in
high-grade tissues (Fig. [186]7h). Collectively, these results
indicated that the presence of aggresomes is likely to be an etiology
of and therapeutic target in certain types of malignancy, including
serous ovarian cancer, in addition to neurodegenerative disorders such
as Parkinson’s disease and dementia with Lewy bodies (DLB)^[187]39.
Fig. 7. FDA-approved MKK7 inhibitor ibrutinib preferentially inhibit
tumorigenicity of OTUD1^high ovarian cancer cells.
[188]Fig. 7
[189]Open in a new tab
a IB analysis of ASK1/JNK pathway proteins derived from OVCAR3 and
SKOV3 cells. b Representative immunofluorescence images of the
aggresome formation in wild-type OVCAR3 and SKOV3 cells. Scale bar,
10 μm. c Representative immunofluorescence images of the aggresome
formation. Immunofluorescence assay was used to evaluate the effect of
OTUD1 depletion on ASK1 aggregation in SKOV3 cells. Scale bar, 10 μm. d
The sphere formation assay was used to examine the effect of IN-8
(2 μg/mL) on spheres formation of OVCAR3 and SKOV3 cells (above panel).
Scale bar, 50 μm. Statistical analysis showing the effect of IN-8 on
number of floating spheres (bottom panel). n = 3. e The sphere
formation assay was used to examine the effect of Selonsertib (1 μM) on
spheres formation of OVCAR3 and SKOV3 cells (above panel). Scale bar,
50 μm. Statistical analysis showing the effect of Selonsertib on number
of floating spheres (bottom panel). n = 3. f SKOV3 cells or OVCAR3 were
subcutaneously injected into BALB/c nude female mice (n = 6 mice per
group) with or without ibrutinib administration (25 mg/kg). After 24 d
or 62 d, the mice were sacrificed, tumors were collected and their
volume was measured. Tumor growth curve of indicated xenografts are
used to reflect the tumor volume. g IB analysis of the JNK pathway
proteins (JNK/p-JNK, c-Jun/c-Jun) derived from xenografted tumors with
or without ibrutinib treatment. h Immunofluorescence assay was used to
investigate the correlation of OTUD1 protein level and ASK1
distribution in clinical serous ovarian cancer tissues. Scale bar,
10 μm. I A proposed model of this study. P values are calculated using
unpaired Student’s t test (d, e), two-way ANOVA (f). n.s. not
significant. Representative of n = 3 independent experiments (a–c, e,
g). Source data are provided as a Source Data file.
Discussion
By screening for key components in tumorigenesis and CSC maintenance in
ovarian cancer, we identified the deubiquitinase OTUD1 as a unique
factor and found that its high expression predicts poor prognosis and
potentiates ovarian tumor cell stemness maintenance (Fig. [190]1).
OTUD1 physically interacts with ASK1 and is involved in MAPK pathway
regulation, which is essential for the maintenance of CSC properties
(Fig. [191]2). Although OTUD1 is capable of stabilizing the ASK1
protein, its deubiquitinase activity appears to be unnecessary for this
effect. Interestingly, the OTUD1 protein clearly forms proteasomal
degradation-resistant aggresomes in the cytoplasm and recruits ASK1 to
these aggresomes via protein interactions, therefore significantly
stabilizing ASK1 while maintaining ASK1 activity (Fig. [192]3). A
peptide located in the intrinsically disordered OTUD1 N-terminal region
is responsible for OTUD1 aggresome formation and associated ASK1
aggregation, as well as downstream JNK activation (Fig. [193]4).
Consistent with these findings, the abrogation of OTUD1 aggresome
formation efficiently inhibited ovarian cancer cell stemness
(Fig. [194]5). ASK1/JNK inhibitors very effectively suppressed OSCS
induced by OTUD1 overexpression (Fig. [195]6), and ibrutinib, an
FDA-approved MKK7 inhibitor, preferentially reduced the tumorigenicity
of OTUD1^high ovarian cancer cells (Fig. [196]7). Based on the above
observations, we proposed the following model. OTUD1 is more likely to
be overexpressed in high-grade serous ovarian cancer tissues, and its
overexpression is sufficient for aggresome-like organelle formation via
its N-terminal intrinsically disordered region. The resultant aggresome
sequesters ASK1 and stabilizes it, thereby activating JNK signaling and
significantly promoting tumor cell stemness. Disruption of aggresome
formation or treatment with ibrutinib might be a promising therapeutic
strategy for OTUD1^high ovarian cancer by targeting CSCs
(Fig. [197]7h).
Aggresome formation in the cytosol is a general response of mammalian
cells when the capacity of the proteasome is exceeded by the production
of misfolded proteins^[198]40. Because the accumulation of misfolded
proteins is cytotoxic, aggresomes are thought to be cytoprotective, and
the misfolded proteins are stored or eventually degraded by autophagy.
The presence of aggresomes has long been thought to be a hallmark of
neurodegenerative diseases; one example is the formation of cytoplasmic
TDP-43 aggregates in amyotrophic lateral sclerosis (ALS) and a subtype
of frontotemporal lobar degeneration (FTLD)^[199]41. While some
proteins have been shown to be more substantially complexed/aggregated
in cancer tissues than in normal tissues, a comprehensive understanding
of the pathological role of aggresomes in human cancer is still
lacking. The presence of aggresomes has been shown to be correlated
with poor prognosis in cancer patients^[200]42. A previous study showed
that structurally destabilized p53 mutants could coaggregate with
wild-type p53 and its paralogs p63 and p73, thus blocking the
transcriptional activity and proapoptotic function of wild-type
p53^[201]43,[202]44. In contrast, our results indicated that instead of
inactivating the sequestered ASK1 protein, OTUD1 stabilizes it and
maintains or improves its ability to activate the downstream JNK
pathway, most likely because proteins incorporated in aggresomes
usually escape from proteasome-dependent degradation. This process is
precisely controlled, as ASK1 aggregation is tightly coupled with OTUD1
aggresome formation (Fig. [203]3), suggesting that aggresomes in tumor
cells could promote cancer progression via distinct mechanisms, namely,
by either eliminating the tumor suppressor activity of sequestered
proteins or protecting targeted proteins from being degraded in a
proteasome-dependent manner.
Deubiquitinases remove posttranslational ubiquitin (Ub) modifications
from proteins and regulate nearly all Ub-dependent processes^[204]45.
Previous cancer research involving the OTUD1 protein has mainly focused
on its canonical deubiquitinase activity. For example, OTUD1 can
inhibit colonic inflammation by deubiquitinating RIPK1 to suppress
NF-κB signaling^[205]46. OTUD1 can deubiquitinate SMAD7 to enable
SMURF2 binding and subsequent TβRI turnover on the cell surface to
increase breast cancer metastasis^[206]47. OTUD1 was also shown to
exacerbate colon cancer progression by deubiquitinating and stabilizing
iron-responsive element-binding protein 2 (IREB2)^[207]48, indicating
that OTUD1 could act as an oncogene. On the other hand, a recent study
showed that OTUD1 could noncanonically regulate AKT activity through
its disordered N-terminal region^[208]49, a mechanism that has been
much less appreciated, probably because OTUD1 lacks homology with any
known secondary structure domains, indicating that the exact function
of the intrinsically disordered N-terminus of OTUD1 is unknown and
remains to be further addressed. In this study, our results clearly
showed that high OTUD1 expression is closely correlated with poor
prognosis and that a region spanning amino acids 105–164 is capable of
mediating OTUD1 self-assembly both in vitro and in vivo, an event that
is needed for OTUD1-associated aggresome formation, ASK1 stabilization,
tumor cell stemness maintenance and tumorigenesis, underscoring the
importance of the disordered OTUD1 N-terminal region in the regulation
of characteristic cancer signaling pathways and reinforcing the idea
that OTUD1 is an oncogene in ovarian cancer.
The signaling pathways that regulate the stemness maintenance and
survival of CSCs have become targets for cancer therapy. Previous
studies have established that the activated JNK signaling pathway
contributes to CSC maintenance^[209]30; however, the molecular
mechanism by which JNK is activated in OCSCs is not well understood.
Our results indicated that a high level of OTUD1 drives JNK pathway
activation, suggesting that OTUD1 might serve as a biomarker for JNK
inhibition-based OCSC-targeted therapy. In addition, numerous JNK
inhibitors have been developed, but very few such drugs are available
for clinical use; one of the major reasons for this lack is their
intolerable cytotoxicity. We found that the FDA-approved drug
ibrutinib, which has been used for treating relapsed/refractory
nongerminal center B-cell-like diffuse large B-cell lymphoma (DLBCL) in
clinical trials^[210]50, was recently reported to be an MKK7
inhibitor^[211]38, and we found that it effectively suppresses OSCS and
tumorigenesis in OTUD1^high ovarian cancer and sensitizes cancer cells
to chemotherapy. Given that CSCs are mostly arrested in G0 phase and
are relatively static, thus evading the cytotoxic effect of
chemotherapeutic drugs^[212]51, our work may provide a rationale for
developing a safe and reliable combination treatment for ovarian
cancer.
In summary, our work suggests that aggresome formation in tumor cells
could function as a signaling hub and that aggresome-based therapy has
translational potential for patients with OTUD1^high ovarian cancer. A
previous study showed that a conserved aggregation-nucleating sequence
within the hydrophobic core of the DNA-binding domain of p53 becomes
exposed after mutation and is responsible for mutant p53 self-assembly.
Moreover, a peptide designed to inhibit p53 amyloid formation to rescue
p53 functions and reduce tumorigenesis and metastasis was also
reported, prompting us to explore strategies such as targeting OTUD1
and abrogating its aggresome formation, which warrants further
investigation.
Methods
Study approval
All the animal used in this study were evaluated and approved by the
Experimental Animal Welfare Ethics Committee, Zhongnan Hospital of
Wuhan University (license no. ZN2022255). Tumor tissues from patients
were obtained from the Shanghai Outdo Biotech Company. The use of
tissue microarray for research purposes was approved by the Ethics
Committee of Shanghai Outdo Biotech Company (license no. YBM-05-02).
Written informed patient consent was obtained prior to the commencement
of the study. All procedures performed in the study were in accordance
with the Declaration of Helsinki.
Cell culture
293T (SCSP-502), SKOV3 (TCHu185), CAOV3 (SCSP-570), and OVCAR3
(TCHu228) cell lines originated from the National Collection of
Authenticated Cell Cultures (Shanghai, China). OVCAR8 cell line was a
gift from Professor Chaoyang Sun (Tongji Hospital, Tongji Medical
College, Huazhong University of Science and Technology). Cell lines
were authenticated by STR profiling and verified to be mycoplasma
negative using the MycoBlue Mycoplasma Detector kit (Vazyme, China,
#D101-01). 293T, SKOV3 and CAOV3 cell lines were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Hyclone, America, #SH30243.01)
supplemented with 10% fetal bovine serum (FBS, AusgeneX, Australia,
#FBS500-S). OVCAR8 cell line was cultured in RPMI-1640 (Hyclone,
America) supplemented with 10% FBS. OVCAR3 cell line was cultured in
RPMI-1640 supplemented with 20% FBS. SKOV3, CAOV3, OVCAR8 and OVCAR3
cell line were cultured in DMEM/F12 (Gibco, America, #11320033)
supplemented with 2% B-27 supplement (Gibico, America, #12587010),
20 ng/mL epidermal growth factor (EGF, Solarbio, China, [213]P00033),
20 ng/mL basic fibroblast growth factor (bFGF, Solarbio, China,
#[214]P00032), and 5 μg/mL insulin (Solarbio, China, #I8040) for the
tumor sphere formation assay.
Lentiviral transduction
Lentiviruses were produced by transfecting 293T cells with
sgRNA-targeting plasmids, phage plasmids, or pCW-EGFP plasmids, along
with the packaging plasmids pMD2.G and psPAX2. The cell supernatants
were harvested 48 h after transfection using Lipofectamine 2000 reagent
(Invitrogen, America, #11668019) and were used to infect SKOV3, OVCAR8,
CAOV3 or 293T cells, respectively. To obtain stable cell lines, cells
were infected at low confluence (20%) for 24 h with lentiviral
supernatants diluted 1:1 with normal culture medium in the presence of
5 ng/mL of polybrene (Solarbio, China, #H8761). Forty-eight hours after
infection, stable cell lines were obtained by using puromycin
(Solarbio, China, #P8230) for one week. The maintenance concentration
of puromycin was 2 µg/mL for SKOV3 cells, OVCAR3, OVAR8, and CAOV3
cells.
Vectors and plasmids production
Wild type, truncating mutation and deletion mutation of OTUD1, as well
as wild type, truncating mutation of ASK1, were cloned into pHAGE for
mammalian expression with different tags. Additionally, the
pET28a-mEGFP-OTUD1, pET28a-mEGFP -Δ105–164 constructs were cloned for
protein purification. OTUD1 C320A point mutations were generated by
site-directed mutagenesis PCR from the pHAGE-Flag-OTUD1 plasmids.
Antibodies
Anti-OTUD1 (HPA038504, 1:1000) and Anti-OTUD1 (HPA038503, 1:200) were
purchased from Atlas Antibodies. Anti-OTUD1 (29921-1-AP, 1:200),
anti-ASK1 (67072-1-Ig, 1:1000), Anti-ERK (11257-1-AP, 1:1000), anti-p38
(114064-1-AP, 1:1000), anti-JNK, (66210-1-Ig, 1:1000), anti-HSP70,
(10995-1-AP, 1:200), anti-P62 SQSTM1, (18420-1-AP, 1:200), anti-GAPDH,
(60004-1-Ig, 1:3000), and anti-Flag tag, (20543-1-AP, 1:1000) were
purchased from Proteintech. Anti-phospho-p44/42 MAPK (Erk1/2)
Thr202/Tyr204) (#9101, 1:1000), anti-phospho- p38 MAPK (Thr180/Tyr182)
(#9211, 1:1000), anti-SAPK/JNK (Phospho-Thr183/Tyr185) (81E11) (#4668,
1:1000), anti-c-Jun (Phospho-Ser73) (D47G9), (#3270, 1:1000), and
anti-c-Jun (#9165, 1:1000) were purchased from Cell Signaling
Technology. Anti-ASK1 (380952, 1:1000) was purchased from. Anti-ASK1
(Phospho-Thr838) (orb335764, 1:1000) was purchased from Biorbyt.
anti-HA tag (2063, 1:1000) and anti-Myc tag (2097, 1:1000) were
purchased from DiaAn Biotech. Goat anti-Rabbit IgG H&L (Alexa Fluor®
488) (ab15007, 1:200), Goat anti-Rabbit IgG H&L (Alexa Fluor 555)
(ab150078, 1:200), APC Mouse Anti-Human CD44(G44-26) (559942, 1:50),
and FITC Mouse Anti-Human CD133(W6B3C1) (567029, 1:50) were purchased
from BD Pharmingen. Goat anti- Rabbit IgG H&L (HRP) (BF03008, 1:5000)
and Goat anti-Mouse IgG H&L (HRP) (BF03001, 1:5000) were purchased from
Biodragon,
CRISPR/Cas9 generation of OTUD1 Knockout
The CRISPR-Cas9-based editing of OTUD1 was generated by cloning the
single guide RNAs (sgRNAs) into a lentiCRISPR V2 vector, which encodes
both Cas9 and a sgRNA of interest. The sgRNA design and cloning were
conducted according to the general cloning protocols of the Feng Zhang
lab ([215]https://zlab.bio/guide-design-resources) and the sequences of
sgRNAs were listed in Supplementary Table [216]1. All the constructs
were validated by DNA sequencing and details of the plasmid
constructions are available upon request. Cells were plated on 6-well
plates the day before the transfection. Transfection was performed
using Lipofectamine 2000 (Invetrogen, # 11668019) according to the
manufacturer’s instructions. A total of 2 μg of sgRNA plasmid DNA, 1 μg
of plasmid pMD2.G, and 1 μg of plasmid psPAX per dish with 2:3
Lipofectamine 2000 ratio were used. Supernatant containing virus was
collected 48 h after transfection.
RNA extraction and qRT-PCR
Total RNA was extracted using TRIzol (Invitrogen, America, #15596018)
and thenreverse transcribed using All-in-One cDNA synthesis SuperMix
(Bimake, America, #[217]B24403). qPCR was carried out using SYBR Green
(Bimake, America, #[218]B21802) using CFX Connect Real Time PCR
Detection System (Bio-Rad, America). All target gene-expression levels
were normalized to GAPDH. The primer sequences are provided in
Supplementary Table [219]2.
Western blotting
Cells were lysed with 100 μL RIPA lysis buffer (Beyotime, China,
#P0013K) containing protease inhibitor mixture (MedChemExpress,
America, #HY-K0010) for 30 min at 4 °C followed by ultrasonic-assisted
extraction. The cell lysates were then sonicated and centrifuged to
obtain the supernatant. 20–40 μg of proteins were separated by
SDS-PAGE. Primary antibodies and secondary anti-mouse or anti-rabbit
antibodies conjugated to horseradish peroxidase were then incubated
with the blots. Protein bands were visualized on X-ray film (Sigma,
America) using an ECL recognition system (Vazyme, China, #SQ201).
Antibodies used are shown in Supplementary Table [220]3.
Co-immunoprecipitation
The cell pellet was resuspended in ice-cold cell lysis buffer and
incubated on ice for 30 min. The cells were then sonicated in an ice
bath and centrifuged at 12,000 × g at 4 °C for 10 min. The supernatant
was transferred to a fresh tube, and the remaining lysates were
incubated with the lysis buffer treated at room temperature for 40 min.
Immunoprecipitation was performed with anti-FLAG magnetic beads
(MedChemExpress, America, #HY-K0207) or with anti-HA magnetic beads
(MedChemExpress, America, #HY-K0201) for 6–8 h at 4 °C. Proteins
immobilized on the beads were eluted with 2× loading buffer and heated
at 100 °C for 10 min. Protein samples were resolved by SDS-PAGE using
8–12% acrylamide gels, transferred to nitrocellulose (NC) membranes,
and immunoblotted using the indicated antibodies.
Gel-shift assay
Cells were lysed with RIPA lysis buffer (Beyotime, China, #P0013K)
containing protease inhibitor mixture (MedChemExpress, America,
#HY-K0010) for 30 min at 4 °C, followed by ultrasonic-assisted
extraction. The cell lysates were sonicated and centrifuged to obtained
the supernatant. Part of the supernatants were collected and heated at
100 °C. The heated and unheated proteins were then separated by
SDS-PAGE. Coomassie Brilliant Blue dye was used to stain the SDS-PAGE
gels to visualize proteins.
RNA sequencing
Total RNA was extracted from different groups using TRIzol (Invitrogen,
America, #15596018). The quality of RNA was measured, and RNA
sequencing were performed by Novogene Biotech company (Beijing, China).
The cDNA libraries were sequenced using the Illumina novaseq-PE150
sequencing platform at Novogene Biotech company (Beijing, China). For
the downstream analyses with RNA-Seq data, the Featurecount software
was used to quantitatively analyze the gene level. The EdgeR software
was used to analyze the differential expression of gene in each sample.
To obtain the Kyoto Encyclopedia of Genes and Genomes (KEGG) result,
the KOBAS softewarewas employed.
CCK-8 assay
Cell viability was measured using the CCK-8 kit (MedChemExpress,
America, #HY-K0301). Cells were seeded at 30–50% density in 96-well
plates. Then, after 0 h, 24 h, 48 h, and 72 h of incubation, all medium
was removed and replaced with the fresh medium containing CCK-8 reagent
for 1 h.Tthe plates were then read by micro-plate reader (BioTeK,
America) at 450 nm.
Colony formation assay
Ovarian cancer cells were seeded into 6-well plates. When the cells
were adherent, they were stimulated with appropriate inhibitors, such
as SP600125 (MedChemExpress, America, #HY-12041), ibrutinib
(MedChemExpress, America, # HY-10997), IN-8(MedChemExpress, America,
#HY13319), selonsetib (MedChemExpress, America, #HY18938), T-5224
(MedChemExpress, America, #HY-12270) and cisplatin (MedChemExpress,
America, # HY-17394) or a combination of both for 2-3 weeks. Then, the
medium was removed and fixation solution was added into the plates at
room temperature for 10 min. The colony cells were soaked in 0.5%
crystal violet solution and incubated at room temperature for 2 h after
removing the fixation solution.
Transwell invasion assays
Thaw the stock 10 mL Matrigel® (Corning, America, #356234) overnight at
4 °C by placing inside at cold room. Dilute Matrigel at a 1:8 ratio
with chilled serum-free growth medium before coating. 100 μL the
chilled diluted Matrigel was placed directly onto the center of the
Transwell insert. Place the plates in a humidified incubator at 37 °C
for 60 min to allow gelling. Meanwhile, a proper density of cancer
cells in 200 ml serum-free growth medium were added to the upper
chamber of replicate and 500 μL serum-containing medium was added to
the lower chamber. The plate was incubated for 24–72 h at 37 °C. The
inserts were stained with crystal violet and were observed under
microscope (Olympus, America, IX53 + DP80) for identification.
Soft agar colony formation assay
1% noble agar and 0.6% noble agar were prepared. The bottom layer was
obtained by mixing 1% noble agar with equal volume of 2X cell culture
medium. Once the lower layer of agar solidified, the upper layer
containing with cells were plated onto lower layer. The cells and agar
mixture were placed into a 37 °C cell culture incubator for around 21
days. Formed colonies were stained with 0.01% crystal violet and
photographs of wells were obtained by using microscope (Olympus,
America, IX53 + DP80).
Tumor sphere formation assays
Single-cell suspensions of ovarian cancer cells (4 × 10^2 cells/mL)
were plated on ultra-low attachment 24 wells plates (Corning, America)
and cultured in phenol red-free DMEM/F12 (Gibco, America) containing
B27 supplement (Gibico, America, #12587010) and 20 ng/mL epidermal
growth factor (EGF, Solarbio, China, #[221]P00033), 20 ng/mL basic
fibroblast growth factor (bFGF, Solarbio, China, #[222]P00032), and
5 μg/mL insulin (Solarbio, China, #I8040). Tumorsphere were visualized
under a phase-contrast microscope (Olympus, America, IX53 + DP80).
Tissue microarray immunohistochemistry assay
Tissue chips containing with 4 cases of low grade ovarian serous
carcinoma and 54 cases of high grade ovarian serous carcinoma were
obtained from Shanghai Outdo Biotech Company (#HOvaC070PT01). The
slides were deparaffinized in xylene for 10 min and then rinsed with
distilled water. After that, the slides were treated with citrate
buffer and microwaved for 10 min. Once cooled, the slides were rinsed
with TBST for 3 min each. Subsequently, the slides were exposed to 1%
H[2]O[2] for 10 min, followed by another rinse with distilled water.
The slides were then blocked in 5% BSA for 1 h. The tissues chips were
incubated with the primary antibody overnight at 4 °C, and then in the
secondary antibody for 1 h, as listed in Supplementary Table [223]2.
Finally, the indicated proteins were visualized using the microscope
(Olympus, America, #IX53 + DP80). The glass slides were scanned using a
panoramic scanning instrument, and the staining results were analyzed
using CaseViewer software. The used of tissue microarray for research
purposed was approved by the Ethics Committee of Shanghai Outdo Biotech
Company (license no. YBM-05-02).
Immunofluorescence assay
In the case of cells, ovarian cancer cells or 293T were seeded on cover
glass. The cells were fixed by 4% paraformaldehyde and permeabilized
with 0.2% triton X-100. The cover glass was then rinsed and blocked in
5% BSA for 10 min. Subsequently, the glass slides were incubated with
primary antibodies (listed in Supplementary Table [224]2) with
different reactivity overnight at 4 °C. The slides were then incubated
in the fluorescent-dye conjugated secondary antibodies, washed with
PBST, and stained with DAPI (4’6-diamidino-2-phenylindole, Beyotim,
China, #P0131) for 5 min for nucleus labeling. After air-drying for
10 min, the samples were observed using a confocal microscope (Leica,
Germany, #TCS SP8).
In the case of tissues, firstly, formalin-fixed paraffin-embedded
tissue sections were dewaxed and rehydrated. Next, the tissue sections
were incubated with the primary antibody for 8 h at room temperature
following heat-induced antigen retrieval. Finally, the tissue sections
were incubated with a fluorophore-conjugated secondary antibody
according to the manufacturer’s instructions. The tissue sections were
observed using confocal microscope (Leica, Germany, #TCS SP8).
Fluorescence recovery after photo-bleaching measurements
Confocal fluorescence measurements were taken using a Leica inverted
microscope with a confocal laser scanning module. EGFP fluorescence was
monitored at 488 nm. Each Fluorescence Recovery After Photo-Bleaching
measurements (FRAP) assay started with a baseline control image of 5
cells, followed by 1 s photobleaching of the region of interest using
high-intensity 488 nm illumination with >90% EGFP fluorescence.
Typically, images were recorded at normal laser intensity after
photobleaching.
Xenograft assay
5-week-old female BALB/c nude mice were used as wild-type and acquired
from Wuhan Wanqianjiaxing Biotechnology Co., Ltd (China, Wuhan). All
animals were maintained in a specific pathogen-free environment and
housed with no more than five animals per cage under 12 light/12 dark
cycle, temperatures of 22 ± 2 °C with 50 ± 10% humidity.
In the xenotransplantation limiting dilution assay, different stable
SKOV3 cells (empty vector, OTUD1^WT, OTUD1^△105–164) were prepared for
xenograft assay. 5-week-old female BALB/c nude mice were acquired from
Wuhan Wanqianjiaxing Biotechnology Co., Ltd (China, Wuhan). After a
week of adjustable feeding, the mice were divided into 3 groups (18
mice each group). Cells were serially diluted to obtain the following
final cell concentrations: dilution group 1 was 1 × 10^4 cells/each;
dilution group 2 was 1 × 10^5 cells/each; dilution group 3 was 1 × 10^6
cells/each. Different stable SKOV3 cells were injected into the left or
right armpit flank subcutaneously. Tumor incidence and size were
monitored within the indicated days after injection. All mice are
euthanized by cervical dislocation after deep anesthesia. After all
mice were sacrificed, subcutaneous tumors were further tissue analyzed.
The permitted maximal tumor size did not exceed 1.5 cm at the largest
diameter.
For SKOV3 cells and OVCAR3 cells, 5-week-old female BALB/c nude female
mice (Wuhan Wanqianjiaxing Bioscience (China)) were used. At 6th week,
SKOV3-sgCtrl or SKOV3-sgOTUD1 cells were randomly injected into the
left or right armpit flank subcutaneously, 5 × 10^6 cells per needle.
At 6th week, OVCAR3-sgCtrl or OVCAR3-sgOTUD1 cells were randomly
injected into the left or right armpit flank subcutaneously, 1 × 10^7
cells per needle. Tumor incidence and size were monitored within the
indicated days after injection. The permitted maximal tumor size did
not exceed 1.5 cm at the largest diameter. All mice are euthanized by
cervical dislocation after deep anesthesia. After all mice were
sacrificed, subcutaneous tumors were further tissue analyzed.
For SKOV3 cells, 5-week-old female BALB/c nude female mice (Wuhan
Wanqianjiaxing Bioscience (China)) were used. At 6th week, 1 × 10^7
SKOV3 cells were randomly injected into the left or right armpit flank
subcutaneously. On day 14 post-injection, the mice were randomly
divided into 2 groups and treated with ibrutinib (25 mg/kg,
MedChemExpress, America, #HY-10997) or vehicle control (20% SBE-β-CD in
saline) each other day by intraperitoneal injection. Tumor incidence
was monitored within indicated days after injection. All mice are
euthanized by cervical dislocation after deep anesthesia. The tumor
tissues were harvested and used to detect the levels of OTUD1 related
proteins. For OVCAR3 cells, the same method was used for xenograft
assay in BALB/c nude female mice, and the inoculation site was the left
or right armpit flank subcutaneously. On day 45 after injection, the
mice were randomly divided into 2 groups and treated with ibrutinib
(25 mg/kg, MedChemExpress, America, #HY-10997) or vehicle control (20%
SBE-β-CD in saline) each other day by intraperitoneal injection. All
mice are euthanized by cervical dislocation after deep anesthesia.
Tumor incidence was monitored on indicated days after injection. For
OVCAR8 cells, 1 × 10^7 OVCAR8 cells were injected subcutaneously into
6-week-old female BALB/c nude female mice, and the inoculation site was
the left or right armpit flank subcutaneously. On day 24
post-injection, the mice were randomly divided into 2 groups and
treated with ibrutinib (25 mg/kg, MedChemExpress, America, #HY-10997)
or vehicle control (20% SBE-β-CD in saline) each other day by
intraperitoneal injection. Tumor incidence and size were monitored
within the indicated days after injection. All mice are euthanized by
cervical dislocation after deep anesthesia. After all mice were
sacrificed, subcutaneous tumors were further tissue analyzed. The
permitted maximal tumor size did not exceed 1.5 cm at the largest
diameter.
Statistics
All graphs in this study were generated by GraphPad Prism 9.
Statistical tests were shown in the Figure legends. Data are presented
as mean ± standard error (SD) unless otherwise stated. Two-tailed
Student’s t test (unpaired), one-way ANOVA test or two-way ANOVA test
was used to assess the significance of the experiments. P value < 0.05
was considered to be statistically significant.
Reporting summary
Further information on research design is available in the [225]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[226]Supplementary Information^ (4.5MB, pdf)
[227]Peer Review File^ (816.1KB, pdf)
[228]41467_2024_45698_MOESM3_ESM.pdf^ (90.8KB, pdf)
Description of Additional Supplementary Files
[229]Supplementary Data 1-2^ (761.7KB, xlsx)
[230]Reporting Summary^ (201.4KB, pdf)
Source data
[231]Source Data^ (5.1MB, xls)
Acknowledgements