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SENP1 promotes p27kip1 nuclear export though enhanced SUMOylation in cholangiocarcinoma leading to increased cell proliferation and chemoresistance

  • Authors:
    • Kainian Jiang
    • Wei Yang
    • Jie Huang
    • Xiaolong Tan
    • Yan Liu
    • Saiya Tu
    • Jian Luo
  • View Affiliations / Copyright

    Affiliations: Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China, Department of Hepatobiliary and Pancreatic Surgery, The Third People's Hospital of Hubei Province, Wuhan, Hubei 430030, P.R. China, Second Clinical College, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
    Copyright: © Jiang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 141
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    Published online on: July 11, 2025
       https://doi.org/10.3892/ijmm.2025.5582
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Abstract

SUMOylation is a critical post‑translational modification, serving as a key role in nucleocytoplasmic translocation, transcriptional cofactor stabilization and modulation of chromatin remodeling factors, which are associated with oncogenesis, tumor progression and chemotherapy resistance in various types of cancer. SUMOylation was performed by small ubiquitin‑like modifier (SUMO), a kind of small ubiquitin‑like modifier, which was attached or removed from the substrates. The excessive export of nuclear p27kip1 induced by SUMOylation is associated with cell proliferation and chemotherapy resistance in cholangiocarcinoma (CCA). However, the exact underlying mechanism remains currently unknown. The present study investigated SUMO specific peptidase 1 (SENP1), which is known to participate in SUMOylation by activating nuclear SUMO1 precursors and deSUMOylating cytoplasmic substrates. SENP1 exhibited increased expression levels in CCA specimens compared with that in adjacent non‑cancerous tissues, as confirmed by bioinformatics analysis and immunohistochemical assays. A significant correlation between SENP1 and p27kip1 expression levels was observed. SENP1 overexpression significantly increased cytoplasmic p27kip1 expression levels, thereby promoting CCA cell proliferation, accelerating the G1‑S cell cycle transition and reducing chemical sensitivity through increasing overall SUMOylation of p27kip1, as confirmed via western blotting, immunofluorescence, flow cytometry, Cell Counting Kit‑8, 5‑ethynyl‑2'‑deoxyuridine incorporation and SUMOylation tests. By contrast, SENP1 knockdown demonstrated the opposite results. Subsequently, the use of ML‑792, COH000 and leptomycin B treatments, and the mutant variant SENP1‑C603A demonstrated that SENP1 regulates the functionality of p27kip1 through nuclear SUMOylation rather than cytoplasmic deSUMOylation. The involvement of SENP1 represents a pivotal role in governing the nucleocytoplasmic shuttling of p27kip1. SENP1 knockdown could effectively impede CCA cell proliferation and enhance the chemosensitivity of cis‑platinum by modulating the nuclear export of p27kip1 through SUMOylation, thus offering a potential therapeutic approach for CCA in the future.
View Figures

Figure 1

Analysis of TCGA data and clinical
samples demonstrating the gene and protein SENP1 expression levels
in CCA tissues compared with that in normal tissue samples. (A)
Analysis of TCGA data demonstrated increased SENP1 gene expression
in numerous types of cancer evaluated using the UALCAN software.
The red box indicates the CCA data. (B) TCGA data analysis showed
increased gene expression levels of SENP1 CCA, which were
associated with lymph node metastasis and tumor stage, evaluated
using the UALCAN software. (C) IHC analysis demonstrated increased
SENP1 protein expression levels in the CCA cells compared with
those of adjacent healthy tissues. The red arrows indicate nuclei
with a different expression level of SENP1. Scale bar, 25
µm; magnification, ×40. (D) Correlation analysis
demonstrated a significant positive association between SENP1 and
p27kip1 expression levels in CCA tissues using GEPIA 2 analysis
(r=0.61; P<0.01). (E) IHC analysis demonstrated that the
expression of SENP1 and the cytoplasmic p27kip1 localization
increase with increasing CCA tumor stages. Error bar, mean ± SEM;
scale bar, 25 µm; magnification, ×40. *P<0.05,
**P<0.01 and ***P<0.001. ns, not
significant; CCA, cholangiocarcinoma; SENP1, SUMO specific
peptidase 1; TCGA, The Cancer Genome Atlas; TPM, transcripts per
million; IHC, immunohistochemical.

Figure 2

SENP1 knockdown or overexpression in
CCA cell lines. SENP1 knockdown or overexpression did not affect
localization or expression levels of p27kip1. (A) Localization of
SENP1 in RBE, HuCCT1 and TFK1 CCA cell lines. Immunofluorescence
assays showed predominant nuclear localization of SENP1 with
limited cytoplasmic presence in all CCA cell lines. (B) Western
blotting demonstrated the effect of siSENP1 1-3 in the RBE cell
line; SENP1 knockdown did not affect p27kip1 expression levels
(siGAPDH was used as the positive control to confirm that the
transfection reagent was effective). (C) Western blotting
demonstrated the effects of SENP1-oe in the RBE cell line; SENP1-oe
did not affect p27kip1 expression levels. (D) Subcellular
localization of SENP1 is unaffected in siSENP1 cells, compared with
that in VEC cells. (E) Subcellular localization of SENP1 is
unaffected in SENP1-oe cells, compared with that in VEC cells.
Error bar, mean ± SEM; scale bar, 30 µm; magnification,
×100. **P<0.01, ***P<0.001 and
****P<0.0001. CCA, cholangiocarcinoma; SENP1, SUMO
specific peptidase 1; NC, negative control; VEC, empty vector
lentivirus; si, small interfering; oe, overexpression.

Figure 3

SENP1 influences p27kip1 SUMOylation
and subcellular localization. (A) Interactions between p27kip1 and
SENP1 co-IP assay demonstrated using co-IP assays. (B) SENP1-oe
promoted the SUMOylation of p27kip1 in RBE cells. (C) Western
blotting and the nuclear and cytoplasmic proteins isolation assays
showed the increased nuclear p27kip1 and decreased cytoplasmic
p27kip1 expression levels in the RBE cells. (D) Immunofluorescence
showed increased nuclear p27kip1 and decreased cytoplasmic p27kip1
levels in the RBE cells. (E) Time-dependent p27kip1 expression
levels in the nucleus and cytoplasm within SENP1-oe cells. (F)
Time-dependent p27kip1 SUMOylation levels within SENP1-oe cells.
(G) Transfection with siSENP1 reduced the SUMOylation of total
p27kip1 in the RBE cells. (H) Western blotting and the nuclear and
cytoplasmic proteins isolation assays showed the reduction of
nuclear p27kip1 and increase of cytoplasmic p27kip1 in the RBE
cells. (I) Immunofluorescence assay showed decreased nuclear
p27kip1 and increased cytoplasmic p27kip1 levels in the RBE cells.
(J) Time-dependent p27kip1 expression levels in the nucleus and
cytoplasm. (K) Time-dependent SUMOylation levels of p27kip1
following SENP1 transfection. Error bar, mean ± SEM; scale bar, 30
µm; magnification, ×100. *P<0.05,
**P<0.01, ***P<0.001 and
****P<0.0001. SENP1, SUMO specific peptidase 1; NC,
negative control; VEC, empty vector lentivirus; si, small
interfering; oe, overexpression; IP, immunoprecipitation.

Figure 4

SUMOylation levels of nuclear and
cytoplasmic p27kip1 in LMB-treated cells. (A) LMB treatment
restricted the p27kip1 inside the nucleus of SENP1-oe RBE cells.
(B) LMB-treated RBE cells showed significantly increased levels of
nuclear SUMOylated p27kip1, but no significant changes in
cytoplasmic SUMOylated p27kip1 expression levels. (C) LMB treatment
restricted the p27kip1 inside the nucleus of siSENP1 RBE cells. (D)
LMB-treated siSENP1 RBE cells showed significantly decreased levels
of nuclear SUMOylated p27kip1, but no significant changes in
cytoplasmic SUMOylated p27kip1 expression levels. Error bar, mean ±
SEM. **P<0.01, ***P<0.001,
****P<0.0001 and *****P<0.00001. ns,
not significant; SENP1, SUMO specific peptidase 1; VEC, empty
vector lentivirus; si, small interfering; oe, overexpression; LMB,
leptomycin B.

Figure 5

Hydrolase activity of SENP1 serves a
crucial role in p27kip1 nuclear export compared with isopeptidase
activity. (A) The flow chart shows the full pathway of SUMOylation
where the SUMO precursors were firstly hydrolyzed by SENP1 to be
matured and subsequently catalyzed by SUMO E1, E2 and E3 to
conjugate to the substrates, which were finally deconjugated by
SENP1. The red circle shows SENP1 hydrolyzed SUMO precursor and
deconjugated SUMO-substrate in the pathway of SUMOylation. (B)
ML-792 and COH000-treated RBE cells showed decreased SUMOylation
levels of p27kip1 similar to that of siSENP1 cells. (C)
ML-792-treated RBE cells showed a similar distribution of p27kip1
between the nucleus and cytoplasm compared with that of siSENP1
cells. (D) Western blot assays showed the SENP1-C603A was
successfully transfected in the cells. (E) Immunofluorescence
assays showed that SENP1-C603A promoted p27kip1 cytoplasmic
retention. (F) Western blot assays showed that SENP1-C603A promoted
the p27kip1 nuclear export similar to SENP1-oe cells. (G)
Cytoplasmic SUMOylated p27kip1 of SENP1-C603A shows no significant
difference compared with SENP1-oe cells. Error bar, mean ± SEM;
scale bar, 30 µm; magnification, ×100.
*P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001. GG,
glycine-glycine; ctrl, control; ns, not significant; SENP1, SUMO
specific peptidase 1; VEC, empty vector lentivirus; si, small
interfering; oe, overexpression.

Figure 6

Redistribution of p27kip1 mediated by
SENP1-regulated SUMOylation influences the cell cycle and
proliferation rates of RBE cells. (A) An IP assay showed SENP1-oe
transfection reduced the interactions between p27kip1 and CDK2 or
cyclin E1. An IP assay showed SENP1 knockdown promoted interactions
between p27kip1 and CDK2 or cyclin E1 in RBE cells. (B) SENP1-oe
and -C603A mutation promoted G1-S phase transition as
assessed using flow cytometry analysis in RBE cells. (C) siSENP1
transfection reduced G1-S phase transition as assessed
via flow cytometry analysis in RBE cells. (D) A Cell Counting Kit 8
assay showed that the SENP1-oe and -C603A mutation promoted cell
proliferation, while siSENP1 inhibited proliferation of RBE cells.
(E) An EdU assay showed SENP1-oe and -C603A mutation increased cell
proliferation compared with VEC cells, while siSENP1 decreased
proliferation in RBE cells, compared with that in NC cells. Scale
bar, 300 µm; magnification, ×10. Error bar, mean ± SEM.
*P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001. ns, not
significant; SENP1, SUMO specific peptidase 1; VEC, empty vector
lentivirus; NC, negative control; si, small interfering; oe,
overexpression; IB, immunoblotting; IP, immunoprecipitation; EdU,
5-ethynyl-2′-deoxyuridine.

Figure 7

Redistribution of p27kip1 mediated by
SENP1-regulated SUMOylation affects chemotherapy resistance in RBE
cells. (A) The IC50 of RBE cells against cis-platinum
was reduced in siSENP1 cells, while SENP1-oe cells increased the
IC50 of RBE cells against cis-platinum, compared with
that of VEC cells (B) SENP1-oe and -C603A cells showed increased
expression levels of chemotherapy resistance-associated proteins
MDR1 and MRP1. (C) SENP1-oe and -C603A cells showed decreased
expression levels of apoptotic proteins Bax and cleaved caspase-3
(normalized to caspase-3), and increased expression levels of
anti-apoptotic protein Bcl-2, with 4 µM cis-platinum
treatment. (D) siSENP1 cells showed decreased expression of
chemotherapy resistance-associated proteins MDR1 and MRP1. (E)
siSENP1 cells showed increased expression levels of apoptotic
proteins Bax and cleaved caspase-3 (normalized to caspase-3), and
decreased expression levels of anti-apoptotic protein Bcl-2 under 4
µM cis-platinum. (F) siSENP1 cells showed an increased
apoptotic rate with 4 µM cis-platinum treatment analyzed by
flow cytometry, and SENP1-oe cells showed a decreased apoptotic
rate compared with that of NC cells. Error bar, mean ± SEM.
*P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001. SENP1,
SUMO specific peptidase 1; VEC, empty vector lentivirus; NC,
negative control; si, small interfering; oe, overexpression; MDR1,
multidrug resistance 1; MRP1, multidrug resistance-associated
protein 1.
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Copy and paste a formatted citation
Spandidos Publications style
Jiang K, Yang W, Huang J, Tan X, Liu Y, Tu S and Luo J: SENP1 promotes p27kip1 nuclear export though enhanced SUMOylation in cholangiocarcinoma leading to increased cell proliferation and chemoresistance. Int J Mol Med 56: 141, 2025.
APA
Jiang, K., Yang, W., Huang, J., Tan, X., Liu, Y., Tu, S., & Luo, J. (2025). SENP1 promotes p27kip1 nuclear export though enhanced SUMOylation in cholangiocarcinoma leading to increased cell proliferation and chemoresistance. International Journal of Molecular Medicine, 56, 141. https://doi.org/10.3892/ijmm.2025.5582
MLA
Jiang, K., Yang, W., Huang, J., Tan, X., Liu, Y., Tu, S., Luo, J."SENP1 promotes p27kip1 nuclear export though enhanced SUMOylation in cholangiocarcinoma leading to increased cell proliferation and chemoresistance". International Journal of Molecular Medicine 56.3 (2025): 141.
Chicago
Jiang, K., Yang, W., Huang, J., Tan, X., Liu, Y., Tu, S., Luo, J."SENP1 promotes p27kip1 nuclear export though enhanced SUMOylation in cholangiocarcinoma leading to increased cell proliferation and chemoresistance". International Journal of Molecular Medicine 56, no. 3 (2025): 141. https://doi.org/10.3892/ijmm.2025.5582
Copy and paste a formatted citation
x
Spandidos Publications style
Jiang K, Yang W, Huang J, Tan X, Liu Y, Tu S and Luo J: SENP1 promotes p27kip1 nuclear export though enhanced SUMOylation in cholangiocarcinoma leading to increased cell proliferation and chemoresistance. Int J Mol Med 56: 141, 2025.
APA
Jiang, K., Yang, W., Huang, J., Tan, X., Liu, Y., Tu, S., & Luo, J. (2025). SENP1 promotes p27kip1 nuclear export though enhanced SUMOylation in cholangiocarcinoma leading to increased cell proliferation and chemoresistance. International Journal of Molecular Medicine, 56, 141. https://doi.org/10.3892/ijmm.2025.5582
MLA
Jiang, K., Yang, W., Huang, J., Tan, X., Liu, Y., Tu, S., Luo, J."SENP1 promotes p27kip1 nuclear export though enhanced SUMOylation in cholangiocarcinoma leading to increased cell proliferation and chemoresistance". International Journal of Molecular Medicine 56.3 (2025): 141.
Chicago
Jiang, K., Yang, W., Huang, J., Tan, X., Liu, Y., Tu, S., Luo, J."SENP1 promotes p27kip1 nuclear export though enhanced SUMOylation in cholangiocarcinoma leading to increased cell proliferation and chemoresistance". International Journal of Molecular Medicine 56, no. 3 (2025): 141. https://doi.org/10.3892/ijmm.2025.5582
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