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CD44 knockdown and TGF‑β inhibition modulate cell proliferation and invasion in claudin‑low breast cancer cells

  • Authors:
    • Ryoichi Matsunuma
    • Kazuo Kinoshita
    • Sae Imada
    • Shoko Sato
    • Ryosuke Hayami
    • Michiko Tsuneizumi
  • View Affiliations / Copyright

    Affiliations: Department of Breast Surgery, Shizuoka General Hospital, Shizuoka, Shizuoka 420‑8527, Japan, Evolutionary Medicine, Shizuoka Graduate University of Public Health, Shizuoka, Shizuoka 420‑0881, Japan
    Copyright: © Matsunuma et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 7
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    Published online on: October 30, 2025
       https://doi.org/10.3892/or.2025.9012
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Abstract

CD44 serves a dual role in supporting tumor survival and promoting invasion. Claudin‑low breast cancer, characterized by a CD44+/CD24‑ phenotype and epithelial‑mesenchymal transition (EMT), displays aggressive behavior. The present study investigated the interaction between CD44 and TGF‑β signaling, and assessed the cellular effects of their combined inhibition. CD44 was knocked down in claudin‑low breast cancer cell lines (SUM159 and MDA‑MB‑231), and the TGF‑β receptor (TGFBR) inhibitor LY2109761 (LY‑61) was applied for treatment. Cell viability (MTT assay), apoptosis (annexin V assay), invasion (Transwell assay), colony formation and Smad2 phosphorylation (western blotting) were evaluated. CD44 knockdown reduced viability and increased apoptosis but did not markedly suppress invasion. Although TGF‑β stimulation enhanced Smad2 phosphorylation, CD44 knockdown alone did not increase Smad2 activation, indicating that it does not directly regulate Smad2. However, LY‑61 inhibited TGF‑β‑induced Smad2 phosphorylation, effectively counteracting pro‑invasive signaling. Notably, while CD44 knockdown alone had a negligible impact on invasion, its combination with LY‑61 markedly reduced the invasive capacity and colony formation of cells compared with the control (control cells transduced with non‑targeting short hairpin RNA without LY‑61 treatment). LY‑61 induced S phase accumulation, which was more pronounced in SUM159 cells than in MDA‑MB‑231 cells, indicating cell line‑specific effects on cell‑cycle regulation. Clinical data indicated that low CD44 expression was associated with improved survival in patients with claudin‑low breast cancer, despite its potential to enhance EMT signaling. These findings suggested that CD44 knockdown enhanced the response to TGFBR inhibition. Although CD44 depletion may increase EMT‑related signaling, invasion was primarily suppressed by TGF‑β blockade, and the combination with CD44 knockdown further enhanced the inhibition of proliferative phenotypes compared with either treatment alone. This dual‑targeting approach warrants further investigation in claudin‑low breast cancer.
View Figures

Figure 1

CNAs and mutation burden across
breast cancer subtypes (METABRIC dataset). (A) Bar plots showing
the distribution of CNAs for TGFBR1, TGFBR2, CD44, BRCA1 and BRCA2
across breast cancer subtypes (claudin-low, luminal A, luminal B,
Her2-enriched, basal and normal-like) based on the METABRIC
dataset. CNAs were categorized as deletions (blue), diploid
(orange), gains (gray) and amplifications (yellow). (B) Histogram
representing the distribution of mutation counts in claudin-low
(n=179) and basal (n=197) breast cancer samples based on the
METABRIC dataset. Claudin-low tumors exhibited a significantly
lower mutational burden (mean ± SD, 4.43±2.70) compared with basal
tumors (mean ± SD, 6.39±3.84) (Mann-Whitney U test; P<0.0001).
CNA, copy number alteration; TGFBR, TGF-β receptor.

Figure 2

Kaplan-Meier survival curves for
overall survival based on CD44 expression across breast cancer
subtypes (METABRIC dataset). Kaplan-Meier curves showing overall
survival based on CD44 expression levels (high, blue; low, red)
across breast cancer subtypes. Statistical comparisons were
performed using the log-rank test. Significant differences in
survival were observed in the claudin-low (P=0.0419) and luminal B
(P=0.0053) subtypes. No statistically significant differences were
noted in the basal, luminal A and HER2-enriched subtypes.

Figure 3

Additive antitumor effects of LY-61
and CD44 knockdown in claudin-low breast cancer cell lines. (A)
Relative viability of breast cancer cell lines (SUM159, MDA-MB-231
and MDA-MB-468) following treatment with TGF-β receptor inhibitors
(LY-61, Ly3200882, SB431542 and Galunisertib; all at 10 µM). Cells
(1,000 per well) were plated in 96-well plates, and drugs were
added 24 h after plating. Cell viability was measured using the MTT
assay on day 5 after plating. Statistical comparisons were made
using one-way ANOVA followed by Tukey's multiple comparisons test
for each cell line. (B) Western blot analysis of CD44 expression in
SUM159 and MDA-MB-231 cells following shRNA transduction.
Densitometric semi-quantification of CD44 protein levels relative
to GAPDH is shown below the blots (n=3). Data are presented as the
mean ± SD. Statistical comparisons were performed using unpaired
two-tailed t-tests. (C) Relative viability of MDA-MB-231 and SUM159
cells following CD44 knockdown (shCD44) and/or treatment with LY-61
(10 µM). Cells (1,000 per well) were plated in 96-well plates, and
LY-61 was added 24 h after plating. Cell viability was determined
using an MTT assay on day 5 after plating. Statistical significance
was assessed using two-way ANOVA followed by Tukey's multiple
comparisons test. (D) Colony formation assays showing relative
colony numbers in MDA-MB-231 and SUM159 cells following CD44
knockdown and/or LY-61 treatment. Statistical comparisons were
performed using two-way ANOVA followed by Tukey's multiple
comparisons test. (E) Invasion assays in SUM159 and MDA-MB-231
cells showing the relative absorbance and representative microscopy
images of invaded cells stained on the underside of basement
membrane matrix-coated inserts. Cells were treated with LY-61
and/or subjected to CD44 knockdown. Statistical analysis was
performed using two-way ANOVA followed by Tukey's multiple
comparisons test. Representative images are shown at a
magnification of ×100. (A and C-E) Data are presented as the mean ±
SD of three independent experiments (n=3). *P<0.05; **P<0.01;
***P<0.001; ****P<0.0001. Ctrl, control; LY-61, LY2109761;
NS, not significant; sh/shRNA, short hairpin RNA.

Figure 4

Effects of LY-61 and CD44 knockdown
on the TGF-β/Smad2 pathway in claudin-low breast cancer cell lines.
(A) Co-immunoprecipitation assays showing that CD44 co-precipitated
with TGFBR1 in MDA-MB-231 cells. The CD44-TGFBR1 interaction signal
was reduced in shCD44 cells. No detectable co-precipitation was
observed between CD44 and TGFBR2. (B) Western blotting and RT-qPCR
showing Smad2 protein and mRNA levels in MDA-MB-231 and SUM159
cells following CD44 knockdown and/or LY-61 treatment.
Representative western blots (top), densitometric
semi-quantification of Smad2 protein levels (middle) and mRNA
expression levels determined by RT-qPCR (bottom) are shown. Data
are presented as the mean ± SD from three independent experiments
(n=3). (C) Western blot analysis of p-Smad2 and total Smad2 protein
levels after TGF-β stimulation in MDA-MB-231 and SUM159 cells with
or without CD44 knockdown. The semi-quantification of Smad2
phosphorylation was performed by calculating the p-Smad2/Smad2
ratio. CD44 knockdown increased TGF-β-induced p-Smad2 levels in
both cell lines. (D) Western blotting showing the effect of LY-61
treatment on p-Smad2 levels in MDA-MB-231 and SUM159 cells.
Densitometric semi-quantification of the p-Smad2/Smad2 ratio from
three independent experiments (n=3) is shown below the blots. (E)
Relative mRNA expression levels of Snai1 and Twist in
SUM159 and MDA-MB-231 cells following CD44 knockdown and/or LY-61
treatment, as determined using RT-qPCR. Data are presented as the
mean ± SD from three independent experiments (n=3). Statistical
significance was assessed using one-way ANOVA followed by (B)
Dunnett's or (D and E) Tukey's multiple comparisons test, or (C)
unpaired two-tailed Student's t-test. *P<0.05; **P<0.01.
Ctrl, control; LY-61, LY2109761; NS, not significant; p-,
phosphorylated; RT-qPCR, reverse transcription-quantitative PCR;
sh, short hairpin RNA; Snai1, snail family transcriptional
repressor 1; TGFBR, TGF-β receptor; IP, immunoprecipitation.

Figure 5

Effects of LY-61 and CD44 knockdown
on cell cycle progression and apoptosis in claudin-low breast
cancer cells. (A and B) Flow cytometry analysis of cell cycle
distribution in (A) SUM159 and (B) MDA-MB-231 cells treated with
LY-61 and/or CD44 knockdown. LY-61 treatment induced S phase
accumulation, most prominently in SUM159 cells. In SUM159 cells,
CD44 knockdown increased G2 phase accumulation,
suggesting delayed progression through the cell cycle. By contrast,
in MDA-MB-231 cells, the proportion of cells in the G2
phase remained relatively unchanged. The tables summarize the
percentages of cells in each phase. Percentages may not total
exactly 100% due to the exclusion of sub-G1 and debris
populations and rounding errors (values were rounded to one decimal
place after calculation from two decimal places, which may cause
minor deviations such as totals slightly >100%). (C and D)
Apoptosis analysis using annexin V/7-AAD staining in (C) SUM159 and
(D) MDA-MB-231 cells following CD44 knockdown and/or LY-61
treatment. CD44 knockdown increased apoptosis, whereas LY-61
treatment alone had a minimal effect. 7-AAD, 7-aminoactinomycin D;
Ctrl, control; LY-61, LY2109761; sh, short hairpin RNA.
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Copy and paste a formatted citation
Spandidos Publications style
Matsunuma R, Kinoshita K, Imada S, Sato S, Hayami R and Tsuneizumi M: CD44 knockdown and TGF‑&beta; inhibition modulate cell proliferation and invasion in claudin‑low breast cancer cells. Oncol Rep 55: 7, 2026.
APA
Matsunuma, R., Kinoshita, K., Imada, S., Sato, S., Hayami, R., & Tsuneizumi, M. (2026). CD44 knockdown and TGF‑&beta; inhibition modulate cell proliferation and invasion in claudin‑low breast cancer cells. Oncology Reports, 55, 7. https://doi.org/10.3892/or.2025.9012
MLA
Matsunuma, R., Kinoshita, K., Imada, S., Sato, S., Hayami, R., Tsuneizumi, M."CD44 knockdown and TGF‑&beta; inhibition modulate cell proliferation and invasion in claudin‑low breast cancer cells". Oncology Reports 55.1 (2026): 7.
Chicago
Matsunuma, R., Kinoshita, K., Imada, S., Sato, S., Hayami, R., Tsuneizumi, M."CD44 knockdown and TGF‑&beta; inhibition modulate cell proliferation and invasion in claudin‑low breast cancer cells". Oncology Reports 55, no. 1 (2026): 7. https://doi.org/10.3892/or.2025.9012
Copy and paste a formatted citation
x
Spandidos Publications style
Matsunuma R, Kinoshita K, Imada S, Sato S, Hayami R and Tsuneizumi M: CD44 knockdown and TGF‑&beta; inhibition modulate cell proliferation and invasion in claudin‑low breast cancer cells. Oncol Rep 55: 7, 2026.
APA
Matsunuma, R., Kinoshita, K., Imada, S., Sato, S., Hayami, R., & Tsuneizumi, M. (2026). CD44 knockdown and TGF‑&beta; inhibition modulate cell proliferation and invasion in claudin‑low breast cancer cells. Oncology Reports, 55, 7. https://doi.org/10.3892/or.2025.9012
MLA
Matsunuma, R., Kinoshita, K., Imada, S., Sato, S., Hayami, R., Tsuneizumi, M."CD44 knockdown and TGF‑&beta; inhibition modulate cell proliferation and invasion in claudin‑low breast cancer cells". Oncology Reports 55.1 (2026): 7.
Chicago
Matsunuma, R., Kinoshita, K., Imada, S., Sato, S., Hayami, R., Tsuneizumi, M."CD44 knockdown and TGF‑&beta; inhibition modulate cell proliferation and invasion in claudin‑low breast cancer cells". Oncology Reports 55, no. 1 (2026): 7. https://doi.org/10.3892/or.2025.9012
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