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Article

Molecular signatures of triple‑negative breast cancer cells acquiring palbociclib resistance via continuous exposure

  • Authors:
    • Daichi Enomoto
    • Yumika Iwasa
    • Natsuho Irie
    • Haruki Ohata
    • Yuki Uemichi
    • Miyuki Mabuchi
    • Tadashi Shimizu
    • Kohji Takara
  • View Affiliations / Copyright

    Affiliations: Faculty of Pharmaceutical Sciences, Hyogo Medical University, Kobe, Hyogo 650‑8530, Japan
  • Article Number: 405
    |
    Published online on: July 10, 2026
       https://doi.org/10.3892/ol.2026.15760
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Abstract

Palbociclib, a cyclin‑dependent kinase (CDK)‑4/6 inhibitor, exhibits therapeutic potential for triple‑negative breast cancer (TNBC), for which effective treatments remain limited. However, understanding the mechanisms underlying drug resistance is essential for its clinical application. Although the mechanisms underlying resistance to CDK4/6 inhibitors in hormone receptor‑positive breast cancer have been investigated, the mechanisms by which continuous drug exposure induces resistance in TNBC cells remain unclear. Therefore, the present study aimed to establish palbociclib‑resistant TNBC cells using a continuous drug exposure model and to elucidate their characteristics. MDA‑MB‑231 cells, a human TNBC cell type with wild‑type retinoblastoma (Rb) protein, were continuously exposed to palbociclib at gradually increasing concentrations (0.01‑1 µM) to establish resistant cells, which were named MB231/PalR cells. Drug sensitivity was subsequently evaluated using cell viability assays; gene and protein expression levels were analyzed by quantitative polymerase chain reaction and western blotting, respectively; and P‑glycoprotein‑mediated efflux capacity was evaluated using rhodamine 123 staining with a flow cytometer. Compared with parental cells, MB231/PalR cells exhibited reduced sensitivity to palbociclib and abemaciclib. Rb protein expression levels were decreased, without affecting RB1 mRNA expression, in MB231/PalR cells. The expression levels of CCNE1, CDK6, ATP‑binding cassette (ABC)B1 mRNA and cyclin E1 protein were increased, whereas those of ABCG2 and CD274 mRNA were decreased in resistant cells. Furthermore, MB231/PalR cells exhibited reduced sensitivity to the CDK2 inhibitor CVT‑313, and co‑treatment with CVT‑313 failed to restore palbociclib sensitivity. Although P‑glycoprotein efflux capacity and ABCB1 mRNA expression were increased in resistant cells, verapamil treatment did not affect palbociclib sensitivity. In conclusion, continuous exposure of MDA‑MB‑231 cells to palbociclib induced alterations associated with CDK4/6 inhibitor action, resulting in drug resistance. These findings provide mechanistic insights into CDK4/6 inhibitor resistance in TNBC and may support the development of biomarkers and therapeutic strategies to overcome or prevent resistance.
View Figures

Figure 1

Rb expression and phosphorylation in
resistant cells. (A) Western blot analysis of the Rb protein
expression and phosphorylation status. GAPDH was used as a loading
control. Densitometric semi-quantification of western blot images
showing (B) total Rb protein expression, (C) p-Rb protein
expression, and (D) the p-Rb/Rb ratio. Data are presented as
relative values normalized to MDA-MB-231 sample from a
representative experiment. Each bar represents the mean ± standard
error of the mean from three independent biological experiments
(n=3/group). **P<0.01 vs. MDA-MB-231 cells (unpaired Student's
t-test); n.s., not significant. p-, phosphorylated; Rb,
retinoblastoma.

Figure 2

Cyclin E1 expression and effects of
CDK2 inhibition in resistant cells. (A) Western blotting of the
cyclin E1 protein expression (left panels). GAPDH was used as a
loading control. Densitometric semi-quantification of western blot
images. Data are presented as relative values normalized to
MDA-MB-231 sample from a representative experiment (right graph).
Each bar represents the mean ± standard error of the mean from
three independent biological experiments (n=3/group). (B and C)
Cells were treated for 72 h, and sensitivity was evaluated by MTT
assay and IC50 values were calculated. (B) Sensitivity
to the CDK2 inhibitor CVT-313. (C) Sensitivity to palbociclib in
the presence of 5 µM CVT-313. Each bar represents the mean ±
standard deviation (n=3-4/group). **P<0.01 vs. MDA-MB-231 cells
(unpaired Student's t-test). CDK2, cyclin-dependent kinase;
IC50, 50% growth inhibitory concentration.

Figure 3

mRNA expression levels of immune
checkpoint molecules in resistant cells. Relative mRNA levels of
CD274 in MDA-MB-231 and MB231/PalR cells. The Cq values were
used to quantify the PCR products, and relative expression of
CD274 is presented as 2−ΔΔCq. ΔCq was calculated
by subtracting the Cq of ACTB from that of the target gene.
Each bar represents the mean ± standard deviation (n=3/group).
**P<0.01 vs. MDA-MB-231 (unpaired Student's t-test).

Figure 4

Cells with low rhodamine 123
accumulation detected via flow cytometric analysis. (A) Plots are
representative of three independents experiments. (B) Percentages
of cells with low rhodamine 123 accumulation at different exposure
time points. Each bar represents the mean ± standard deviation
(n=3/group). *P<0.05, **P<0.01 (two-way ANOVA followed by the
Tukey's post hoc-test); n.s., not significant. FSC-A, forward
scatter-area.
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Copy and paste a formatted citation
Spandidos Publications style
Enomoto D, Iwasa Y, Irie N, Ohata H, Uemichi Y, Mabuchi M, Shimizu T and Takara K: Molecular signatures of triple‑negative breast cancer cells acquiring palbociclib resistance via continuous exposure. Oncol Lett 32: 405, 2026.
APA
Enomoto, D., Iwasa, Y., Irie, N., Ohata, H., Uemichi, Y., Mabuchi, M. ... Takara, K. (2026). Molecular signatures of triple‑negative breast cancer cells acquiring palbociclib resistance via continuous exposure. Oncology Letters, 32, 405. https://doi.org/10.3892/ol.2026.15760
MLA
Enomoto, D., Iwasa, Y., Irie, N., Ohata, H., Uemichi, Y., Mabuchi, M., Shimizu, T., Takara, K."Molecular signatures of triple‑negative breast cancer cells acquiring palbociclib resistance via continuous exposure". Oncology Letters 32.3 (2026): 405.
Chicago
Enomoto, D., Iwasa, Y., Irie, N., Ohata, H., Uemichi, Y., Mabuchi, M., Shimizu, T., Takara, K."Molecular signatures of triple‑negative breast cancer cells acquiring palbociclib resistance via continuous exposure". Oncology Letters 32, no. 3 (2026): 405. https://doi.org/10.3892/ol.2026.15760
Copy and paste a formatted citation
x
Spandidos Publications style
Enomoto D, Iwasa Y, Irie N, Ohata H, Uemichi Y, Mabuchi M, Shimizu T and Takara K: Molecular signatures of triple‑negative breast cancer cells acquiring palbociclib resistance via continuous exposure. Oncol Lett 32: 405, 2026.
APA
Enomoto, D., Iwasa, Y., Irie, N., Ohata, H., Uemichi, Y., Mabuchi, M. ... Takara, K. (2026). Molecular signatures of triple‑negative breast cancer cells acquiring palbociclib resistance via continuous exposure. Oncology Letters, 32, 405. https://doi.org/10.3892/ol.2026.15760
MLA
Enomoto, D., Iwasa, Y., Irie, N., Ohata, H., Uemichi, Y., Mabuchi, M., Shimizu, T., Takara, K."Molecular signatures of triple‑negative breast cancer cells acquiring palbociclib resistance via continuous exposure". Oncology Letters 32.3 (2026): 405.
Chicago
Enomoto, D., Iwasa, Y., Irie, N., Ohata, H., Uemichi, Y., Mabuchi, M., Shimizu, T., Takara, K."Molecular signatures of triple‑negative breast cancer cells acquiring palbociclib resistance via continuous exposure". Oncology Letters 32, no. 3 (2026): 405. https://doi.org/10.3892/ol.2026.15760
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