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Inhibition of primary ciliogenesis enhances efficacy of EGFR‑TKIs against non‑small cell lung cancer cells

  • Authors:
    • Liangliang Jin
    • Li Wei
    • Junrui Hua
    • Rong Zhang
    • Jiaxin Chen
    • Jinpeng He
    • Yanli Yang
  • View Affiliations / Copyright

    Affiliations: Department of Pathology, The 940th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army, Lanzhou, Gansu 730050, P.R. China, NHC Key Laboratory of Diagnosis and Therapy of Gastrointestinal Tumor & Clinical Lab, Gansu Provincial Hospital, Lanzhou, Gansu 730000, P.R. China, Key Laboratory of Space Radiobiology of Gansu, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, Gansu 73000, P.R. China, School of Basic Medical Sciences & School of Public Health, Gansu University of Chinese Medicine, Lanzhou, Gansu 730000, P.R. China
    Copyright: © Jin et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 30
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    Published online on: December 4, 2025
       https://doi.org/10.3892/or.2025.9035
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Abstract

Primary cilia are antenna‑like organelles on almost all human cells that sense and transduce extracellular cues into cellular response. Primary cilia have been reported to be implicated in drug resistance in several cancer types, but their roles in cellular response to epidermal growth factor receptor (EGFR)‑tyrosine kinase inhibitors (TKIs) in non‑small cell lung cancer (NSCLC) are still not fully understood. In the present study, it was reported that primary cilia are more prevalent in EGFR‑TKI‑insensitive A549 and H23 cells compared with the drug‑sensitive HCC827 and PC9 cells by immunofluorescence staining assay. Importantly, treatment with EGFR‑TKIs (gefitinib and dacomitinib) results in a dose‑dependent increase in cilia number and length in A549 and H23 cells, an effect not observed in HCC827 and PC9 cells. Upon administration of gefitinib, A549 cells predominantly arrest in the G1 phase detected by flow cytometric analysis, with a minority undergoing cell death and the majority entering senescence. Inhibition of ciliogenesis through the knockdown of IFT88 or ARL13B by targeted small interfering RNAs markedly enhances the sensitivity of A549 cells to EGFR‑TKIs by promoting a shift from senescence to cell death. Furthermore, it was demonstrated by immunoblotting and immunofluorescence colocalization analysis that both the expression and ciliary localization of adenylate cyclase 3 (AC3) are significantly upregulated following EGFR‑TKIs treatment, and the reduction of AC3 expression effectively mitigates cellular drug resistance in A549 cells. These findings highlight a critical role for the cilia‑AC3 axis in modulating cellular response to EGFR‑TKIs, suggesting it as a potential therapeutic target for the treatment of NSCLC.
View Figures

Figure 1

Epidermal growth factor
receptor-tyrosine kinase inhibitor-insensitive A549 cells harbor
high cilia incidence. (A and B) A549 cells were treated with
increasing concentrations of (A) Gef or (B) Dac, and the cell
viability related to untreated control (Ctrl) was measured at 48 h
post-treatment. (C and D) HCC827 cells were treated with increasing
concentrations of (C) Gef or (D) Dac, and the cell viability
related to untreated control was measured at 48 h post-treatment.
(E and F) A549 and HCC827 cells were exposed to increasing
concentrations of Gef for 48 h, and the immunoblotting analysis was
performed to measure the protein expression levels of ERK and p-ERK
at Thr202/Tyr204. α-tubulin (α-tub) was used as a loading control.
(G) Representative images of primary cilia labeled with ARL13B
(red) and basal bodies labeled with γ-tub (green) in A549 and
HCC827 cells, and the cilia incidence was calculated. The nuclei
were counterstained with DAPI (blue). Scale bar, 10 µm. All
quantitative data were obtained from three replicates and shown as
the mean ± SD. Error bars, ± SD. *P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001 compared with 0 µM (0.1% DMSO).
Gef, gefitinib; Dac, dacomitinib; p-, phosphorylated; SD, standard
deviation.

Figure 2

Epidermal growth factor
receptor-tyrosine kinase inhibitors promote ciliogenesis in
dose-dependent manner in A549 but not HCC827 cells. (A)
Representative images of primary cilia labeled with ARL13B (red)
and basal bodies labeled with γ-tub (green) in A549 cells treated
with increasing doses of Gef or Dac for 48 h. (B and C)
Quantification of (B) ciliated cells and the (C) cilium length of
A549 cells treated with 0 (n=117), 1 (n=176), 5 (n=133), 10
(n=216), 25 (n=210), 50 (n=95) µM Gef for 48 h. **P<0.01 and
****P<0.0001 compared with 0 µM; ns, not significant. (D and E)
Quantification of (D) ciliated cells and the (E) cilium length of
A549 cells treated with 0 (n=133), 0.5 (n=153), 1 (n=138), 5
(n=163), 7.5 (n=130), 10 (n=80) µM Dac for 48 h. *P<0.05 and
****P<0.0001 compared with 0 µM; ns, not significant. (F)
Representative images of primary cilia labeled with ARL13B (red)
and basal bodies labeled with γ-tub (green) in HCC827 cells treated
with 0.01 µM Gef or 0.001 µM Dac for 48 h. (G) Quantification of
ciliated cells in HCC827 cells in (F). ns, not significant compared
with DMSO. The nuclei were counterstained with DAPI (blue). Scale
bar, 10 µm. All quantitative data were obtained from three
replicates and shown as the mean ± SD. Error bars, ± SD. Gef,
gefitinib; Dac, dacomitinib; p-, phosphorylated; SD, standard
deviation; ns, not significant.

Figure 3

Gef treatment leads to G1 phase
arrest and senescence. (A and B) Cell cycle distribution assay of
A549 cells treated with DMSO (0.1%) or 25 µM Gef for 1, 2, and 3
days (d) by flow cytometry with (A) PI staining; (B) quantification
data. (C and D) Cell cycle distribution assay of HCC827 cells
treated with DMSO (0.1%) or 0.05 µM Gef for 1, 2, and 3 days by
flow cytometry with (C) PI staining; (D) quantification data. (E)
Immunoblotting analysis of the expression levels of CCNA, CCNB,
CCND, CCNE, CDK1, CDK2, p16 and p21 in A549 cells treated with
DMSO, Gef (25 µM), or Dac (5 µM) for 1 and 3 days. α-tubulin
(α-tub) and GAPDH were used as loading controls. (F) Representative
PI staining images of A549 cells exposed to DMSO (0.1%) or Gef (25
µM) for 1, 2, 3 days, the dead cells (red) were stained with PI and
indicated with red arrows. Scale bar, 100 µm. (G) Immunoblotting
analysis of the expression levels of IFT88, p-ERK (Thr202/Tyr204)
and BCL2 in A549 cells treated with DMSO (0.1%) or Gef (25 µM) for
1, 2, 3 days. α-tubulin (α-tub) was used as a loading control. The
values under the immunoblot bands indicate the quantitative
densitometry value measured using ImageJ software. (H and I) The
(H) cellular senescence assay of A549 cells treated with DMSO
(0.1%) or Gef (25 µM) for 1–3 d labeling by senescence-tracker
(Sen-Tra) fluorescence probe, and the (I) quantification data.
Scale bar, 100 µm. Data are expressed as the mean ± SD. Error bars,
± SD. *P<0.05, **P<0.01 and ***P<0.001 compared with DMSO.
Gef, gefitinib; PI, propidium iodide; CCNA, Cyclin A2; CCNB, Cyclin
B1; CCND, Cyclin D1; CCNE, Cyclin E1; Dac, dacomitinib; BF, bright
field; p-, phosphorylated; SD, standard deviation.

Figure 4

Inhibition of ciliogenesis sensitizes
A549 cells to epidermal growth factor receptor-tyrosine kinase
inhibitors. (A) Immunoblotting analysis of the protein expression
levels of IFT88 and ARL13B in A549 cells transfected with siRNAs
against IFT88 (siIFT88-1/2), ARL13B (siARL13B-1/2), or NC. GAPDH
was employed as a loading control. The values under the immunoblot
bands indicate the quantitative densitometry value measured using
ImageJ software. (B-D) Immunofluorescence labeling of primary cilia
(ARL13B, red) and basal bodies (γ-tub, green) in A549 control cells
(Ctrl) and cells transfected with siIFT88-1/2, siARL13B-1/2, or NC
following treated with 10 µM Gef for 48 h, (C) the proportion of
ciliated cells and (D) cilium length in each group. Ctrl, n=52;
Gef-NC, n=59; Gef-siIFT88-1, n=57; Gef-siIFT88-2, n=64;
Gef-siARL13B-1, n=56; Gef-siARL13B-2, n=69. The nuclei were stained
with DAPI (blue). (E and F) A549 cells transfected with NC,
siIFT88-1/2, or ARL13B-1/2 siRNAs were exposed to (E) Gef (0, 5, 10
µM) or (F) Dac (0, 1, 5 µM) for 48 h, the cell viability related to
untreated control was measured. (G and H) A549 cells transfected
with indicated siRNAs were exposed 10 µM Gef for 48 h and stained
with PI to detect the dead cells and quantification data for each
group. Scale bar, 100 µm. Data are expressed as the mean ± SD.
Error bars, ± SD. *P<0.05, **P<0.01 and ***P<0.001
compared with NC. siRNA, small interfering RNA; NC, negative
control; Gef, gefitinib; Dac, dacomitinib; BF, bright field; SD,
standard deviation.

Figure 5

Epidermal growth factor
receptor-tyrosine kinase inhibitors induce AC3 expression and
ciliary localization. (A) Immunoblotting analysis of the expression
levels of AC3 and ARL13B in A549 cells exposed to DMSO (0.1%), Gef
(10 µM) and Dac (5 µM) for 1 and 3 days. (B and C)
Immunofluorescence labeling of primary cilia (Arl13b, green) and
AC3 (red, indicated by white arrows) in A549 cells administrated
with DMSO (0.1%), Gef (10 µM), and Dac (5 µM) for 3 days, and
quantification of AC3 positive (AC3+) cilia in each
group. The nuclei were stained with DAPI (blue). Scale bar, 10 µm.
Data are expressed as the mean ± SD. Error bars, ± SD.
***P<0.001 compared with DMSO. (D) Immunoblotting analysis of
the expression levels of AC3 and ARL13B in HCC827 cells exposed to
DMSO (0.1%), Gef (0.05 µM) and Dac (0.01 µM) for 3 days. (E)
Immunoblotting analysis of the protein expression levels of AC3 and
IFT88 in Gef-treated A549 cells transfected with siIFT88-1
(siIFT88) or NC siRNA for 3 days. (F) Immunofluorescence labeling
of primary cilia (Arl13b, red) and AC3 (green) in cells in (E).
Scale bar, 5 µm. GAPDH was used as a loading control; the values
under the immunoblot bands indicate the quantitative densitometry
value measured using ImageJ software. Gef, gefitinib; Dac,
dacomitinib; SD, standard deviation; si-, small interfering; NC,
negative control; AC3, adenylate cyclase 3.

Figure 6

Silencing AC3 expression enhances
cellular sensitivity to Gef. (A) Immunoblotting analysis of the
expression levels of AC3 and ARL13B in A549 cells transfected with
siRNAs targeting AC3 (siAC3-1/2/3) or NC for 48 h. GAPDH was used
as a loading control. The values under the immunoblot bands
indicate the quantitative densitometry value measured using ImageJ
software. (B) A549 cells transfected NC or siAC3-1/2/3 were exposed
to Gef (0, 5, 10 µM) for 48 h, the cell viability related to
untreated control was measured. (C and D) Immunofluorescence
labeling of primary cilia (Arl13b, red, indicated by white arrows)
in A549 cells transfected with NC and siAC3-1 following
administrated with Gef (10 µM) for 3 days, and quantification of
ciliated cells in each group. The nuclei were stained with DAPI
(blue). Scale bar, 10 µm. Data are expressed as the mean ± SD.
Error bars, ± SD. *P<0.05 and **P<0.01 compared with NC. AC3,
adenylate cyclase 3; Gef, gefitinib; si-, small interfering; NC,
negative control; SD, standard deviation; ns, not significant.
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Copy and paste a formatted citation
Spandidos Publications style
Jin L, Wei L, Hua J, Zhang R, Chen J, He J and Yang Y: Inhibition of primary ciliogenesis enhances efficacy of EGFR‑TKIs against non‑small cell lung cancer cells. Oncol Rep 55: 30, 2026.
APA
Jin, L., Wei, L., Hua, J., Zhang, R., Chen, J., He, J., & Yang, Y. (2026). Inhibition of primary ciliogenesis enhances efficacy of EGFR‑TKIs against non‑small cell lung cancer cells. Oncology Reports, 55, 30. https://doi.org/10.3892/or.2025.9035
MLA
Jin, L., Wei, L., Hua, J., Zhang, R., Chen, J., He, J., Yang, Y."Inhibition of primary ciliogenesis enhances efficacy of EGFR‑TKIs against non‑small cell lung cancer cells". Oncology Reports 55.2 (2026): 30.
Chicago
Jin, L., Wei, L., Hua, J., Zhang, R., Chen, J., He, J., Yang, Y."Inhibition of primary ciliogenesis enhances efficacy of EGFR‑TKIs against non‑small cell lung cancer cells". Oncology Reports 55, no. 2 (2026): 30. https://doi.org/10.3892/or.2025.9035
Copy and paste a formatted citation
x
Spandidos Publications style
Jin L, Wei L, Hua J, Zhang R, Chen J, He J and Yang Y: Inhibition of primary ciliogenesis enhances efficacy of EGFR‑TKIs against non‑small cell lung cancer cells. Oncol Rep 55: 30, 2026.
APA
Jin, L., Wei, L., Hua, J., Zhang, R., Chen, J., He, J., & Yang, Y. (2026). Inhibition of primary ciliogenesis enhances efficacy of EGFR‑TKIs against non‑small cell lung cancer cells. Oncology Reports, 55, 30. https://doi.org/10.3892/or.2025.9035
MLA
Jin, L., Wei, L., Hua, J., Zhang, R., Chen, J., He, J., Yang, Y."Inhibition of primary ciliogenesis enhances efficacy of EGFR‑TKIs against non‑small cell lung cancer cells". Oncology Reports 55.2 (2026): 30.
Chicago
Jin, L., Wei, L., Hua, J., Zhang, R., Chen, J., He, J., Yang, Y."Inhibition of primary ciliogenesis enhances efficacy of EGFR‑TKIs against non‑small cell lung cancer cells". Oncology Reports 55, no. 2 (2026): 30. https://doi.org/10.3892/or.2025.9035
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