Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Dec 2;20(1):270.
doi: 10.1186/s12931-019-1244-2.

Protein tyrosine kinase 2: a novel therapeutic target to overcome acquired EGFR-TKI resistance in non-small cell lung cancer

Xuexia Tong  1   2 , Ryosuke Tanino  1 , Rong Sun  1 , Yukari Tsubata  3 , Tamio Okimoto  1 , Mayumi Takechi  4 , Takeshi Isobe  1
Affiliations

Protein tyrosine kinase 2: a novel therapeutic target to overcome acquired EGFR-TKI resistance in non-small cell lung cancer

Xuexia Tong et al. Respir Res. .

Abstract

Background: Protein tyrosine kinase 2 (PTK2) expression has been reported in various types of human epithelial cancers including lung cancer; however, the role of PTK2 in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) has not been elucidated. We previously reported that pemetrexed-resistant NSCLC cell line PC-9/PEM also acquired EGFR-TKI resistance with constitutive Akt activation, but we could not find a therapeutic target.

Methods: Cell viability in EGFR-mutant NSCLC cell lines was measured by the WST-8 assay. Phosphorylation antibody array assay for receptor tyrosine kinases was performed in PC-9 and PC-9/PEM cell lines. We evaluated the efficacy of EGFR and PTK2 co-inhibition in EGFR-TKI-resistant NSCLC in vitro. Oral defactinib and osimertinib were administered in mice bearing subcutaneous xenografts to evaluate the efficacy of the treatment combination in vivo. Both the PTK2 phosphorylation and the treatment combination efficacy were evaluated in erlotinib-resistant EGFR-mutant NSCLC cell lines.

Results: PTK2 was hyperphosphorylated in PC-9/PEM. Defactinib (PTK2 inhibitor) and PD173074 (FGFR inhibitor) inhibited PTK2 phosphorylation. Combination of PTK2 inhibitor and EGFR-TKI inhibited Akt and induced apoptosis in PC-9/PEM. The combination treatment showed improved in vivo therapeutic efficacy compared to the single-agent treatments. Furthermore, erlotinib-resistant NSCLC cell lines showed PTK2 hyperphosphorylation. PTK2 inhibition in the PTK2 hyperphosphorylated erlotinib-resistant cell lines also recovered EGFR-TKI sensitivity.

Conclusion: PTK2 hyperphosphorylation occurs in various EGFR-TKI-resistant NSCLCs. Combination of PTK2 inhibitor and EGFR-TKI (defactinib and osimertinib) recovered EGFR-TKI sensitivity in the EGFR-TKI-resistant NSCLC. Our study result suggests that this combination therapy may be a viable option to overcome EGFR-TKI resistance in NSCLC.

Keywords: Combined inhibition; Drug resistance; EGFR; PTK2; Tyrosine kinase inhibitor.

PubMed Disclaimer

Conflict of interest statement

Takeshi Isobe reports personal fees from AstraZeneca, personal fees from Pfizer, personal fees from Boehringer Ingelheim, outside the submitted work. Yukari Tsubata reports personal fees from AstraZeneca, personal fees from Daiichi Sankyo, outside the submitted work. Ryosuke Tanino reports grants from Nippon Boehringer Ingelheim, outside the submitted work. The remaining authors declare no competing interest.

Figures

Fig. 1
Fig. 1
The pemetrexed-resistant PC-9 cell line acquired EGFR-TKI resistance. a-e Viabilities of the human EGFR-mutant NSCLC cell line PC-9 and the PEM-resistant cell line PC-9/PEM treated for 72 h with pemetrexed or EGFR-TKI at the indicated concentrations. Data are means (SD), n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t-test
Fig. 2
Fig. 2
PC-9/PEM exhibits PTK2 hyperphosphorylation. a Direct sequencing chromatogram at the T790 site of EGFR in PC-9/PEM. b Copy number quantification of genomic DNA extracted from PC-9, PC-9/PEM, and PC-9/OSI. MET or MAD1L1 copy numbers relative to RPPH1 copy number are shown. MAD1L1 was the negative control. Data are means (SD), n = 3. ***, P < 0.001 by Student’s t-test. c Immunoblots of the phosphorylated and total levels of the indicated proteins in PC-9 and PC-9/PEM cells treated with 1 μM erlotinib (Erl) or DMSO for 96 h. d Expressions of the indicated genes in PC-9 and PC-9/PEM cells. Data are means (SD), n = 3. *, P < 0.05, ***, P < 0.001, ****, P < 0.0001 by Student’s t-test. e Phosphorylation array analysis of 71 phosphorylated human receptor tyrosine kinases extracted from PC-9 and PC-9/PEM cells. f Representative immunoblots of phosphorylated and total PTK2 in PC-9 and PC-9/PEM cells and the ratio of phosphorylated PTK2 to β-actin. Data are means (SD), n = 3. ***, P < 0.001 by Student’s t-test
Fig. 3
Fig. 3
The PTK2 inhibitor defactinib recovers EGFR-TKI sensitivity, but not pemetrexed sensitivity, in PC-9/PEM. a Immunoblots of phosphorylated and total PTK2 in PC-9 and PC-9/PEM cells treated with defactinib at the indicated concentrations for 96 h. b Immunoblots of the phosphorylated and total levels of the indicated proteins in PC-9 and PC-9/PEM cells treated with 3 μM defactinib or DMSO for 96 h. c Viabilities of PC-9 and PC-9/PEM cells treated with defactinib at the indicated concentrations for 96 h. Data are means (SD), n = 3. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Data for treated and untreated cells were compared by one-way ANOVA and Tukey’s HSD multiple comparisons test. d-f Viabilities of PC-9/PEM cells either with or without defactinib and treated with pemetrexed (d), erlotinib (e), or osimertinib (f) at the indicated concentrations for 96 h. Data are means (SD), n = 3. *, P < 0.05; **, P < 0.01. d Data were compared by one-way ANOVA and Games–Howell multiple comparisons test. e and f Data were compared by Student’s t-test
Fig. 4
Fig. 4
EGFR and PTK2 co-inhibition induce apoptosis in PC-9/PEM clone1. a Representative immunoblots of phosphorylated and total PTK2 in PC-9 and PC-9/PEM clone1 cells and the ratio of phosphorylated PTK2 to β-actin. Data are means (SD), n = 3. *, P < 0.05 by Student’s t-test. b Viabilities of PC-9, PC-9/PEM, and PC-9/PEM clone1 cells treated with erlotinib or osimertinib at the indicated concentrations for 96 h. Data are means (SD), n = 3. PC-9 vs. PC-9/PEM, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, PC-9 vs. PC-9/PEM clone1, #, P < 0.05; ##, P < 0.01; ###, P < 0.001; ####, P < 0.0001 by one-way ANOVA and Tukey’s HSD multiple comparisons test. c Viability of PC-9/PEM clone1 cells with or without defactinib and erlotinib (upper) or osimertinib (lower) at the indicated concentrations for 96 h. Data are means (SD), n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data for cells with and without defactinib were compared by Student’s t-test. d Viability of PC-9/PEM clone1 cells with or without 1 μM defactinib and EGFR-TKI (0.01 μM gefitinib, 0.03 μM erlotinib, 0.001 μM afatinib, or 0.01 μM osimertinib). Data are means (SD), n = 3. **, P < 0.01; ****, P < 0.0001, EGFR-TKI + Def vs. Def; ###, P < 0.001; ####, P < 0.0001, NS, not significant by one-way ANOVA and Tukey’s HSD multiple comparisons test. e Immunoblots of the phosphorylated and total levels of the indicated proteins in PC-9 and PC-9/PEM clone1 cells treated with 3 μM defactinib or DMSO for 96 h. f Representative immunoblots of the indicated proteins in PC-9 and PC-9/PEM clone1 cells with or without 1 μM defactinib (Def) and 0.1 μM osimertinib (Osi). Ratios of phospho-Akt to β-actin (upper) and cleaved PARP to β-actin (lower). Data are means (SD), n = 3. **, P < 0.01, ***, P < 0.001, NS, not significant by one-way ANOVA and Tukey’s HSD multiple comparisons test. g Immunoblots of phosphorylated and total PTK2 in PC-9/PEM clone1 cells treated with or without 1 μM defactinib, 1 μM PD173074 or 1 μM BLU-554 for 72 h. h Immunoblots of phosphorylated and total PTK2 in PC-9/PEM clone1 cells transfected with siRNAs against PTK2. The siCtrl was used as a negative control. i Viability of PC-9/PEM clone1 cells transfected with the indicated siRNAs and treated with osimertinib at the indicated concentrations for 96 h. Data are means (SD), n = 3. (siCtrl vs. si#1, *, P < 0.05; **, P < 0.01), (siCtrl vs. si#2, #, P < 0.05; ##, P < 0.01) by one-way ANOVA and Tukey’s HSD multiple comparisons test
Fig. 5
Fig. 5
EGFR-TKI and PTK2 co-inhibition improved therapeutic efficacy in a xenograft model. a Viability of OUS-11 cells treated with 1 μM defactinib and osimertinib at the indicated concentrations for 96 h. Data are means (SD), n = 3. *, P < 0.05; ***, P < 0.001, by Student’s t-test. b Immunoblots of PARP and cleaved PARP in PC-9/PEM clone1 and OUS-11 cells with or without 1 μM defactinib (Def) and 0.1 μM osimertinib (Osi) following long and short exposing time. c and e Tumor volume of xenografts of PC-9 (c) and PC-9/PEM clone1 (e) were measured over time of the treatment after inoculation and the results are shown. Data are means (SD), n = 6. d and f Changes in tumor volumes of xenografts of PC-9 (d) and PC-9/PEM clone1 (f). Data are means (SD), n = 6. g and h Changes in tumor volumes of xenografts at day 30 for PC-9 (g) and PC-9/PEM clone1 (h). Data are means (SD), n = 6. *, P < 0.05; **, P < 0.01, NS, not significant. Groups were compared by one-way ANOVA and Tukey’s HSD multiple comparisons test
Fig. 6
Fig. 6
Defactinib and osimertinib co-treatment inhibited cell proliferation in various EGFR-TKI-resistant cell lines. a Immunoblots of phosphorylated and total PTK2 in PC-9 and PC-9/ER-1-6 cells. b Viabilities of PC-9 and PC-9/ER-4 cells treated with osimertinib at the indicated concentrations for 72 h. Data are means (SD), n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data for treated and untreated cells were compared by Student’s t-test. c Immunoblots of the phosphorylated and total levels of the indicated proteins in PC-9/ER-4 cells treated with defactinib at the indicated concentration for 96 h. d-f Viability of PC-9/ER-4 cells with or without defactinib and osimertinib at the indicated concentrations for 96 h. Data are means (SD), n = 3. **, P < 0.01; ***, P < 0.001, ****, P < 0.0001. Data for untreated cells were compared with those for cells treated with defactinib by one-way ANOVA and Tukey’s HSD multiple comparisons test. Viabilities of PC-9/ER-1 (e) and PC-9/ER-2 (f) cells with or without 1 μM defactinib and osimertinib at the indicated concentrations for 96 h. Data are means (SD), n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t-test
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med. 2008;359:1367–1380. doi: 10.1056/NEJMra0802714. - DOI - PMC - PubMed
    1. Mok TS, Wu Y, Thongprasert S, Yang C, Saijo N, Sunpaweravong P, et al. Gefitinib or carboplatin–paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361:947–957. doi: 10.1056/NEJMoa0810699. - DOI - PubMed
    1. Wu Y-L, Zhou C, Hu C-P, Feng J, Lu S, Huang Y, et al. Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial. Lancet Oncol. 2014;15:213–222. doi: 10.1016/S1470-2045(13)70604-1. - DOI - PubMed
    1. Inoue A, Suzuki T, Fukuhara T, Maemondo M, Kimura Y, Morikawa N, et al. Prospective phase II study of gefitinib for chemotherapy-naïve patients with advanced non-small-cell lung cancer with epidermal growth factor receptor gene mutations. J Clin Oncol. 2006;24:3340–3346. doi: 10.1200/JCO.2005.05.4692. - DOI - PubMed
    1. Riely GJ, Politi KA, Miller VA, Pao W. Update on epidermal growth factor receptor mutations in non-small cell lung cancer. Clin Cancer Res. 2006;12:7232–7241. doi: 10.1158/1078-0432.CCR-06-0658. - DOI - PubMed

MeSH terms