Up-regulation of the Hippo pathway effector TAZ renders lung adenocarcinoma cells harboring EGFR-T790M mutation resistant to gefitinib - PubMed (original) (raw)

Up-regulation of the Hippo pathway effector TAZ renders lung adenocarcinoma cells harboring EGFR-T790M mutation resistant to gefitinib

Wei Xu et al. Cell Biosci. 2015.

Abstract

Background: The T790M mutation of epithelial growth factor receptor (EGFR) is a major cause of the acquired resistance to EGFR tyrosine kinase inhibitor (EGFR-TKIs) treatment for lung cancer patients. The Hippo pathway effector, TAZ, has emerged as a key player in organ growth and tumorigenesis, including lung cancer.

Results: In this study, we have discovered high TAZ expression in non-small cell lung cancer (NSCLC) cells harboring dual mutation and TAZ depletion sensitized their response to EGFR-TKIs. Mechanistically, knockdown of TAZ in T790M-induced resistant cells leaded to reduced anchorage-independent growth in vitro, tumor formation and resistance to gefitinib in vivo, correlated with epithelial-mesenchymal transition (EMT) and suppressed migration and invasion. Furthermore, we confirmed CTGF and AXL, novel EMT markers and potential therapeutic targets for overcoming EGFR inhibitor resistance, as directly transcriptional targets of TAZ.

Conclusions: Taken together, this study suggests that expression of TAZ is an intrinsic mechanism of T790M-induced resistance in response to EGFR-TKIs. Combinational targeting on both EGFR and TAZ may enhance the efficacy of EGFR-TKIs in acquired resistance of NSCLC.

Keywords: EGFR; EMT; Gefitinib; Lung adenocarcinoma; TAZ.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Enhanced expression of TAZ in gefitinib-resistant cell. (A) Lysates derived from human bronchial epithelial cell line 16HBE, lung adenocarcinoma cell lines A549, cisplatin-resistant A549/DDP, gefitinib-sensitive PC9 and gefitinib-resistant PC9/GR were analyzed by western blot using anti-TAZ antibodies. The levels of GAPDH were detected as loading controls. (B) Relative mRNAs of TAZ in lung cancer cell lines were examined by real-time PCR. The endogenous b-actin RNA was used as the internal control. (C) Nuclear fractions in PC9 and PC9/GR cell lines were analyzed by western blot. The levels of histone H3 were detected as loading controls. Densitometric evaluation of TAZ:H3 ratios is illustrated in graph beside. Data are shown as means ± SEM. n=3. Statistical analyses were carried out using Student’s t-test. Significance: * P<0.05. (D) Confocal microscopy of immunofluorescent staining of TAZ in PC9 and PC9/GR cells. Scale bar = 100 mm for original picture and 25 mm for inset. Dapi was used to stain nuclei.

Figure 2

Figure 2

Effect of TAZ on the sensitivity of NSCLC cells to gefitinib. (A) Protein lysate extracted from PC9 cells expressing pEX2-TAZ were subjected to western blot by using anti-TAZ, anti-pAkt, anti-tAkt, anti-pERK and anti-tERK antibodies, respectively. (B) Overexpression of TAZ descreased gefitinib sensitivity in PC9 cells. Cells were transfected and then exposed to various doses of gefitinib for 48 h and viability was accessed by MTT assay as described in Methods. The inhibition rate was calculated according to the following formula: inhibition ratio (%) = [1- A490 (experimental group)/A490 (Control group)] × 100. (C) LY294002, as a inhibition of Akt signaling, abolished TAZ-driven descreased sensitivity to gefitinib of PC9 cell. (D) shRNAs against different regions of TAZ mRNA (shTAZ1 and shTAZ2) were expressed in PC9/GR cells. Expressions of TAZ, pAkt, tAkt, pERK and tERK were detected by western blot. (E) enhanced sensitivity of PC9/GR cells to gefitinib after TAZ knockdown. (F) Enhanced sensitivity to gefitinib after TAZ knockdown were detected in H1975 cells. Procedures and conditions for inhibition rate are described as in (B). Results are expressed as means ± SEM from three independent experiments (*P<0.05). Statistical analyses were carried out using Student’s t-test.

Figure 3

Figure 3

TAZ knockdown in PC9/GR cells suppresses anchorage-independent growth, tumorigenesis and resistant to gefitinib in vivo. (A) Two hundred stable GR-shNC, GR-shTAZ1 and GR-shTAZ2 cells were plated onto 6-well plates and a colony formation assay was assessed and photographed. The rate of colony formation was calculated as follows: colony-forming rate = (average colony number/ plated single cell number) × 100%. The colony-forming rates of GR-shTAZ cells were reduced drastically compared with control group. Results are expressed as means ± SEM from three independent experiments (**P<0.01, ***P<0.001). (B) PC9/GR cells with stable expression of shTAZ2 and shNC were inoculated subcutaneously into right and left flanks of nude mice (n = 6 per group), respectively. Representative photographs of nude mice 29 days after inoculation are shown. (C) Tumor growth curve is indicated. (D) Mice bearing PC9/GR xenografts were treated daily with gefitinib at indicated doses. Data represent means ± SEM (*P<0.05, **P<0.01). Statistical analyses were carried out using Student’s t-test.

Figure 4

Figure 4

TAZ mediates EMT of gefitinib-resistant PC9/GR. (A) TAZ knockdown in PC9/GR cells resulted in a morphology change in PC9/GR by a light microscope. The spindle form of the PC9/GR-shNC cells changed to a round shape. The cell density of the clusters was obviously enhanced in the PC9/GR-shTAZ cells. (B) Cell lysates from PC9/GR and PC9 cells expressing vector, shTAZ and PEX2-TAZ were separated and probed with antibodies for epithelial marker and mesenchymal markers as indicated. TAZ knockdown in PC9/GR cells increased E-cadherin and decreased vimentin, CTGF and AXL. All data were normalized to GAPDH. (C) Luciferase reporter assay in 16HBE cells with TAZ or TAZ-S51A overexpression. 16HBE cells co-transfected with pGL3-CTGF/pGL3-AXL and pEX2-TAZ showed significantly elevated reporter activity compared with controls. Meanwhile, cells co-transfected with pEX2-TAZ-S51A, which disrupts the TEAD binding, exhibited decreased luciferase activity compared with pEX2-TAZ. Cells were harvested 48 h after transfection for measurement of luciferase activity in all experiments. (D) Luciferase reporter assay in PC9/GR cells. Total amounts of DNA and RNA were kept constant. Results are expressed as means ± SEM from three independent experiments. Statistical analyses were carried out using Student’s t-test.

Figure 5

Figure 5

Knockdown of TAZ in PC9/GR suppresses cell migration and invasion. (A) Wound-healing migration assay for PC9/GR cells expressing shNC, shTAZ1 or shTAZ2. The healing of wounds by migrated cells at time 0 and 24 h was imaged. PC9/GR cells expressing shNC migrated faster than PC9/GR cells knocking down TAZ. (B,C) The migration (B) and invasion (C) of control, PC9/GR cells expressing shNC, shTAZ1 or shTAZ2 were assessed by transwell assay and matrigel invasion assay. The migration and invasiveness of TAZ-knocked-down PC9/GR cells decreased 52% - 63% and 53% - 62%, respectively, compared with control cells. Results are expressed as means ± SEM from three independent experiments (** P<0.01, *** P<0.001). Statistical analyses were carried out using Student’s t-test.

Similar articles

Cited by

References

    1. Bell DW, Gore I, Okimoto RA, Godin-Heymann N, Sordella R, Mulloy R, Sharma SV, Brannigan BW, Mohapatra G, Settleman J, Haber DA. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat Genet. 2005;37(12):1315–1316. doi: 10.1038/ng1671. - DOI - PubMed
    1. Yoshida T, Zhang G, Smith MA, Lopez AS, Bai Y, Li J, Fang B, Koomen J, Rawal B, Fisher KJ, Chen AY, Kitano M, Morita Y, Yamaguchi H, Shibata K, Okabe T, Okamoto I, Nakagawa K, Haura EB. Tyrosine Phosphoproteomics Identifies Both Codrivers and Cotargeting Strategies for T790M-Related EGFR-TKI Resistance in Non-Small Cell Lung Cancer. Clin Cancer Res. 2014;20(15):4059–4074. doi: 10.1158/1078-0432.CCR-13-1559. - DOI - PMC - PubMed
    1. Suda K, Onozato R, Yatabe Y, Mitsudomi T. EGFR T790M mutation: a double role in lung cancer cell survival? J Thorac Oncol. 2009;4(1):1–4. doi: 10.1097/JTO.0b013e3181913c9f. - DOI - PubMed
    1. Kim Y, Ko J, Cui Z, Abolhoda A, Ahn JS, Ou SH, Ahn MJ, Park K. The EGFR T790M mutation in acquired resistance to an irreversible second-generation EGFR inhibitor. Mol Cancer Ther. 2012;11(3):784–791. doi: 10.1158/1535-7163.MCT-11-0750. - DOI - PubMed
    1. Mitani A, Nagase T, Fukuchi K, Aburatani H, Makita R, Kurihara H. Transcriptional coactivator with PDZ-binding motif is essential for normal alveolarization in mice. Am J Respir Crit Care Med. 2009;180(4):326–338. doi: 10.1164/rccm.200812-1827OC. - DOI - PubMed

LinkOut - more resources