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CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation

  • Authors:
    • Shujuan Sun
    • Zhenyu Hou
    • Ling Qiang
    • Dongdong Zhou
  • View Affiliations / Copyright

    Affiliations: Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong 250117, P.R. China, Department of Gastrointestinal Surgery, Qilu Hospital, Jinan, Shandong 250012, P.R. China
    Copyright: © Sun et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 208
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    Published online on: September 26, 2025
       https://doi.org/10.3892/ijmm.2025.5649
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Abstract

Centromere protein U (CENPU), a critical component of the kinetochore complex, structurally integrates with spindle microtubules to mediate chromosome segregation during mitosis. However, the association between CENPU expression levels and tumors is largely unknown. Immunohistochemistry and western blotting were used to analyze CENPU expression and prognostic value in breast cancer tissues. CENPU overexpressing/knockdown cell lines were constructed for 4D‑data‑independent acquisition quantitative proteomics and enrichment analyses. Functional assays, including flow cytometry, mammosphere formation, wound healing and Transwell assay, were used to assess the effects of CENPU on breast cancer stemness, migration and invasion. The associations among CENPU, nerve growth factor (NGF), proNGF and furin were also explored through western blotting, co‑immunoprecipitation and ELISA experiments. Finally, xenograft mouse models were established to verify the in vivo effects of CENPU on tumorigenesis and the inhibitory effect of furin inhibitor on CENPU‑promoted tumor growth. In the present study, immunohistochemistry and western blotting assessment of human breast cancer tissue specimens revealed a positive association between CENPU expression and the degree of invasiveness. The aforementioned functional analyses demonstrated that CENPU promoted stem cell‑like behavior and tumorigenicity, and induced malignancy in BC cells. Mechanistically, western blotting analysis demonstrated that CENPU promoted furin activity by inhibiting its lysosomal degradation. Furin, which is a precursor‑processing enzyme of (NGF), promoted the conversion of NGF precursor to NGF, which could promote BC stem cell properties in triple‑negative BC (TNBC). A tumorigenesis assay conducted in xenograft mouse models showed that CENPU promoted tumorigenesis, and treatment with a furin inhibitor suppressed this effect. The findings of the present study revealed that CENPU serves a critical role in furin‑mediated signaling responsible for tumorigenesis. Therefore, CENPU may be a novel molecular target in TNBC.
View Figures

Figure 1

Expression levels of CENPU are
positively associated with the degree of invasiveness in breast
cancer. (A) Immunohistochemical staining and. (B) Western blotting
was performed using four samples of normal breast tissue, four
samples of DCIS tissue and eight samples of IDC tissue. (C)
Quantification of tissue collected from 67 patients (17 patients
with breast dysplasia, 20 patients with DCIS and 30 patients with
IDC) was performed. Scale bar, 50 μm. *P<0.05.
CENPU, centromere protein U; IDC, invasive ductal carcinoma; DCIS,
ductal carcinoma in situ.

Figure 2

CENPU promotes tumorigenesis, stem
cell-like properties, migration and invasion in breast cancer
cells. Western blotting was performed to detect the expression
levels of CD44 and ALDH in (A) MDA-MB-231, (B) Cal51 and (C) T47D
cells with different CENPU expression levels. Association between
CENPU expression and the number of mammospheres per 200 cells in
(E) MDA-MB-231 and Cal51 cells and (G) T47D cells (scale bar, 100
μm). CD24− CD44+ flow cytometry
results showed that CENPU expression levels affected the percentage
of the CD24− CD44+ population among (D)
MDA-MB-231, (F) Cal51 and (H) T47D cells. Transwell assays were
performed to determine the effect of CENPU levels on the migration
and invasiveness of (I) MDA-MB-231, (J) Cal51 and (K) T47D cells
(scale bar, 200 μm). A wound healing assay was performed to
determine the effect of CENPU on the migration potential of (L)
MDA-MB-231, (M) Cal51 and (N) T47D cells (scale bar, 200
μm). *P<0.05; **P<0.01. CENPU,
centromere protein U; sh, short hairpin RNA; APC, allophycocyanin;
ALDH, aldehyde dehydrogenase; PE, phycoerythrin.

Figure 3

CENPU promotes the conversion of
proNGF to NGF. Western blotting experiments were performed to
detect the expression of NGF and proNGF in (A)
MDA-MB-231-shCENPU, (B) Cal51-shCENPU and (C)
T47D-CENPU cells and their corresponding control groups. The
intracellular NGF levels in (D) MDA-MB-231, (E) Cal51 and (F) T47D
cells after 48 h of treatment were analyzed by ELISA using
ultrasonicated cell supernatants. After NGF-neutralizing antibodies
were added to (G) MDA-MB-231-shCENPU, (I)
Cal51-shCENPU and (K) T47D-CENPU cells, and their
corresponding control groups, Transwell migration and invasion
assays were conducted (scale bar, 200 μm). After
NGF-neutralizing antibodies were added to the (H)
MDA-MB-231-shCENPU, (J) Cal51-shCENPU and (L)
T47D-CENPU cells, and their corresponding control groups, a
mammosphere formation assay was performed to measure the number of
mammospheres per 200 cells that formed (scale bar, 100 μm).
*P<0.05; **P<0.01. CENPU, centromere
protein U; sh, short hairpin RNA; NGF, nerve growth factor; proNGF,
NGF precursor; NS, not significant.

Figure 4

CENPU enhances the activity of furin.
(A) Western blotting was performed to determine the expression
levels of furin in MDA-MB-231, Cal51 and T47D cells. (B) Furin
activity in MDA-MB-231, Cal51 and T47D cells was measured based on
fluorescence intensity. (C) Michaelis constant values in the
experimental group and control group of MDA-MB-231, Cal51 and T47D
cells were obtained using double reciprocal plotting. (D)
Mammosphere formation assays were performed after treatment with a
furin inhibitor, and the number of tumor spheres per 200 cells was
quantified (scale bar, 100 μm). **P<0.01.
CENPU, centromere protein U; sh, short hairpin RNA; NS, not
significant; RFU, relative fluorescence units; V, reaction
velocity; S, substrate concentration.

Figure 5

CENPU enhances furin activity by
inhibiting its degradation via the lysosomal pathway. (A) CENPU and
furin were subjected to co-immunoprecipitation. (B) Furin and
proNGF were subjected to co-immunoprecipitation. (C)
MDA-MB-231-shCENPU cells and their corresponding controls
were treated with CHX (20 μM) for the specified duration,
and protein levels were measured by western blotting. (D) Relative
expression of furin. (E) MDA-MB-231 cells were treated with MG132
(10 μM) for 12 h, and then also treated with CHX (20
μM) for the indicated times. Protein levels were detected by
western blotting. (F) Relative expression of furin. (G) MDA-MB-231
cells were treated with CQ (100 μM) for 12 h, and then
treated with CHX (20 μM) for the indicated times. Protein
levels were detected by western blotting. (H) Relative expression
of furin. (I) Cal51-shCENPU cells and their corresponding
controls were treated with CHX (20 μM) for the specified
duration, and protein levels were measured by western blotting. (J)
Relative expression of furin. (K) Cal51 cells were treated with
MG132 (10 μM) for 12 h, and then treated with CHX (20
μM) for the indicated times. Protein levels were detected by
western blotting. (L) Relative expression of furin. (M) Cal51 cells
were treated with CQ (100 μM) for 12 h, and then treated
with CHX (20 μM) for the indicated times. Protein levels
were detected by western blotting. (N) Relative expression of
furin. *P<0.05 vs. DMSO or CON313 group. The
experimental and control groups in panels C-M were run on the same
gel/membrane and incubated with the corresponding primary
antibodies simultaneously. CENPU, centromere protein U; sh, short
hairpin RNA; NS, not significant; proNGF, nerve growth factor
precursor; CHX, cycloheximide; CQ, chloroquine.

Figure 6

CENPU knockdown can inhibit
tumorigenesis. (A) A total of 1×106, 1×105,
1×104 and 1×103 Cal51-shCENPU and
Cal51-CON313 cells were injected into the mammary fat pad of NSG
mice. (B) Sizes of the tumors formed in the Cal51-shCENPU
and Cal51-CON313 groups using different cell concentrations were
measured and statistically analyzed. The tumor tissues collected
from the Cal51-shCENPU and Cal51-CON313 groups were (C)
analyzed by western blotting and (D) subjected to
immunohistochemical staining (scale bar, 50 μm). (E) A total
of 1×106 MDA-MB-231-shCENPU and MDA-MB-231-CON313
cells were injected into the mammary fat pad of nude mice. When the
tumor size was ~100 mm3, the MDA-MB-231-CON313 mice were
administered a furin inhibitor (DECRVKR-CMK) by intraperitoneal
injection. (F) The volume of the final tumor was measured,
indicating that the furin inhibitor significantly inhibited tumor
growth in the MDA-MB-231-CON313 mice. Scale bar, 50 μm.
*P<0.05; **P<0.01;
***P<0.001. CENPU, centromere protein U; sh, short
hairpin RNA; NS, not significant; NGF, nerve growth factor; proNGF,
NGF precursor.

Figure 7

Illustration of the proposed
mechanism by which CENPU promotes triple-negative breast cancer
invasion, tumorigenesis and breast CSC properties through the
furin-proNGF-NGF signaling pathway. CENPU, centromere protein U;
NGF, nerve growth factor; proNGF, NGF precursor; CSC, cancer stem
cell; TrKA, tropomyosin receptor kinase A; CRD, cysteine-rich
domain; JM, juxtamembrane domain.
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Copy and paste a formatted citation
Spandidos Publications style
Sun S, Hou Z, Qiang L and Zhou D: CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation. Int J Mol Med 56: 208, 2025.
APA
Sun, S., Hou, Z., Qiang, L., & Zhou, D. (2025). CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation. International Journal of Molecular Medicine, 56, 208. https://doi.org/10.3892/ijmm.2025.5649
MLA
Sun, S., Hou, Z., Qiang, L., Zhou, D."CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation". International Journal of Molecular Medicine 56.6 (2025): 208.
Chicago
Sun, S., Hou, Z., Qiang, L., Zhou, D."CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation". International Journal of Molecular Medicine 56, no. 6 (2025): 208. https://doi.org/10.3892/ijmm.2025.5649
Copy and paste a formatted citation
x
Spandidos Publications style
Sun S, Hou Z, Qiang L and Zhou D: CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation. Int J Mol Med 56: 208, 2025.
APA
Sun, S., Hou, Z., Qiang, L., & Zhou, D. (2025). CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation. International Journal of Molecular Medicine, 56, 208. https://doi.org/10.3892/ijmm.2025.5649
MLA
Sun, S., Hou, Z., Qiang, L., Zhou, D."CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation". International Journal of Molecular Medicine 56.6 (2025): 208.
Chicago
Sun, S., Hou, Z., Qiang, L., Zhou, D."CENPU promotes tumorigenesis and stem cell properties in triple‑negative breast cancer by suppressing lysosomal furin degradation". International Journal of Molecular Medicine 56, no. 6 (2025): 208. https://doi.org/10.3892/ijmm.2025.5649
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