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Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs
Lung Cancer 73 (2011) 176–182
Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs Jin-Haeng Chung a,1 , Jin Kyung Rho b,1 , Xianhua Xu a , Jong Seok Lee c , Ho Il Yoon c , Choon Taek Lee c , Yun Jung Choi b , Hye-Ryoun Kim b , Cheol Hyeon Kim b , Jae Cheol Lee b,∗ a
Department of Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Gyeonggi-do, Republic of Korea Department of Internal Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological & Medical Science, 215-4, Gongneung-dong, Nowon-gu, Seoul 139-706, Republic of Korea c Department of Internal Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Gyeonggi-do, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 26 May 2010 Received in revised form 22 November 2010 Accepted 23 November 2010 Keywords: Epithelial to mesenchymal transition EGFR Resistance Lung cancer
a b s t r a c t Background: Epithelial-to-mesenchymal transition (EMT), which was related with an acquired resistance to gefitinib, was found in the A549 lung cancer cell line. However, the clinical feasibility of this finding is still questionable. Here, we investigated whether EMT could be detected in a more clinically suitable situation using patient’s tumor and cells with deletion mutation on exon 19 of EGFR gene. Methods: HCC827 cell line was used to establish the subline resistant to EGFR-TKIs. The induction of EMT was analyzed by immunostainings and Western blots in resistant cells and biopsied tissue from a patient with acquired resistance to erlotinib. Migration and invasion assay was performed to characterize the resistant cells. EMT-related genes expression was evaluated by cDNA microarray. Phospho-receptor tyrosine kinase array analysis was carried out to find bypass activating signals such as MET. Results: We found that EMT developed in a lung cancer patient who had an acquired resistance to erlotinib while there were no known resistant mechanisms such as T790M and MET amplification. CL-387,785resistant cells (HCC827/CLR) were obtained by long-term exposure to increasing concentrations of CL387,785 (an irreversible EGFR-TKI). The morphological and molecular maker changes compatible with EMT were also found in HCC827/CLR cells. However, there were also no secondary T790M mutation and MET amplification. Furthermore, the activity of most of tested RTKs including receptor HER family was decreased suggesting that there was no bypass activating signal leading to resistance. These cells showed an enhanced capability for migration (∼1.6-fold) and invasion (∼2.8-fold). Conclusion: EMT should be considered as one of possible mechanisms for the acquired resistance to EGFR-TKIs in lung cancer cells. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction EGFR-TKIs such as gefitinib and erlotinib have been used for the treatment of non-small cell lung cancer and they have shown minimal toxicity and a modest effect [1,2]. Several clinical and molecular factors have been determined to be related with a better response and survival with using these novel drugs [3–5]. Among them, EGFR mutations, including deletion mutations on exon 19 and point mutations on exon 21, have been suggested to be the most reliable predictors [5,6]. Sometimes EGFR-TKIs have shown a rapid and dramatic response for allowing a patient to recover from the impending respiratory failure caused by disseminated lung cancer
∗ Corresponding author. Tel.: +82 2 9701206. E-mail address: [email protected] (J.C. Lee). 1 Both authors contributed equally to this study. 0169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2010.11.011
[7]. However, drug resistance eventually develops in most patients despite the initial good response, and this has limited the median time to progression to almost 1 year [8]. Two major resistance mechanisms have currently been revealed. T790M secondary mutation increases the affinity of the oncogenic mutant EGFR for ATP, and this leads to the reduced efficacy of EGFR-TKIs [9]. Almost half of the patients with acquired resistance seem to have this mutation [10,11]. MET amplification is another mechanism to escape the anti-tumor effect of EGFR-TK inhibitors, which allows cell survival by persistent Akt signaling via bypassing MET signaling when the EGFR signal is blocked in the presence of EGFR-TKI. MET amplification has been found in around 20% of the patients with acquired resistance [12,13]. Through considerable efforts have been made to clarify unknown mechanisms that induce resistance, some other possible suggestions have been presented. Guix et al. demonstrated that activation of insulin-like growth factor receptor (IGFR) signaling
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and the loss of IGF binding proteins (IGFBPs) were involved in the adaptive change of tumor cells for acquiring resistance to gefitinib, although this was not confirmed in any lung cancer cell lines [14]. In addition, we previously reported that epithelial-to-mesenchymal transition (EMT) would contribute to the decreased efficacy of therapy due to the resistance to gefitinib [15]. However, the clinical feasibility of EMT as a mechanism of resistance remains unclear because it was only found in one cell line, A549, which has a K-ras mutation, and this indicates that this cell line is not an appropriate model for acquired resistance to EGFR-TKIs. Therefore in this study, we provide more evidences showing that EMT may contribute the development of resistance during longterm exposure to EGFR-TKIs. 2. Materials and methods 2.1. Cell cultures and reagents HCC827 cells with deletion mutation on exon 19 of EGFR gene were cultured in RPMI 1640 (Invitrogen, Carsbad, CA) that contained 10% fetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin (Invitrogen) at 37 ◦ C in a 5% CO2 atmosphere. CL-387,785 (an irreversible EGFR-TKI) was purchased from Calbiochem (San Diego, CA). 2.2. Establishment of the gefitinib/CL-387,785-resistant cell lines The HCC827/CLR cells were established by using a previously described method [15]. Over a period of 6 months, the HCC827 cells in culture were continuously exposed to increasing concentrations of CL-387,785. The surviving cells were cloned and the CL-387,785resistant cell line was designated as HCC827/CLR. These cell lines can survive exposure to over 100 nM CL-387,785.
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phosphate-buffered saline (PBS) and processed further with using a DAKO EnVision kit (DAKO, Los Angles, CA), as directed by the manufacturer. The color was developed with 3,3 -diaminobenzindine (DAB) that contained 0.3% H2 O2 . Primary antibodies against the following antigens were used: E-cadherin, vimentin and thyroid transcripton factor-1 (TTF-1). 2.7. Fluorescent immunocytochemistry To identify the induction of EMT, immunocytochemical evaluation of E-cadherin and vimentin was performed by using a previously described method [15]. 2.8. cDNA microarray Total RNA was isolated from HCC827 and HCC827/CLR cells using TRIZOL reagent (Invitrogen, Carlsbad, CA), followed by RNeasy Mini Kit (Qiagen, Valencia, CA). For control and test RNAs, the synthesis of target cRNA probes and hybridization were performed using Agilent’s Low RNA Input Linear Amplification kit (Agilent Technology, USA) according to the manufacturer’s instructions. Labeled sample (Cy5 CTP) and reference (Cy3 CTP) were mixed and co-hybridized overnight on the same 44K Agilent’s Human Oligo Microarray. The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technology, Palo Alto, CA). All data normalization and selection of fold-changed genes were performed using GeneSpringGX 7.3 (Agilent Technology). The averages of normalized ratios were calculated by dividing the average of normalized signal channel intensity by the average of normalized control channel intensity.
2.3. MTT assay
2.9. Phospho-receptor tyrosine kinase (RTK) array analysis
Briefly, the cells were seeded onto 96-well plates overnight. Each of the drugs was added in a dose dependent manner and the cells were incubated for 72 h. The viability of the cells was determined by using the MTT assay.
It was performed according to manufacturer’s instructions (R&D Systems, Minneapolis, MN). Briefly, 500 g of lysates was incubated in each membrane. The arrays were washed and incubated with a horseradish peroxidase-conjugated phosphor-tyrosine detection antibody, treated with ECL and exposed to film. Intensity of each spot detected with a phospho-RTK array was measured by ImageQuant software (GE healthcare).
2.4. Immunoblot analysis The membrane was probed with antibody against E-cadherin (DakoCytomation, Carpinteria, CA), -catenin (Cell signaling Technology, Beverly, MA), cytokeratin-8/18 (Santa Cruz Biotechnology, Santa Cruz, CA), vimentin (Calbiochem, San Diego, CA) and -actin (Cell signaling) as the first antibody, and then the membrane was treated with horseradish-peroxidase-conjugated secondary antibody. The membrane was developed using an ECL kit (Amersham Biosciences, Piscataway, NJ). 2.5. Migration and invasion assays The cell migration and invasion assays were done according to a previously described method [15]. The results were expressed as means ± SDs. 2.6. Immunohistochemistry For immunohistochemical analysis, the paraffin sections (4 m thick) were deparaffinized with xylene, rinsed thoroughly with ethanol and then soaked in 0.03% hydrogen peroxide in methanol to inactivate the endogenous peroxidase activity. The sections were incubated with either 10% goat serum or 10% rabbit serum, and then they were covered with the primary antibodies, washed with
3. Results 3.1. EMT was found in a patient with acquired resistance to erlotinib A 50-year-old woman was admitted to Seoul National University Bundang Hospital in August 2006 for the evaluation of a lung mass. Chest CT showed an about 3 cm-sized mass on the right middle lobe of the lung without enlargement of the mediastinal lymph nodes (Fig. 1A). The acinar type of adenocarcinoma was diagnosed by percutaneous fine needle aspiration biopsy. However, disseminated tiny nodules were found on the pleura during the operation, and this indicated the advanced stage of her disease. Because the family of our patient strongly wanted to remove the mass although the status of her disease was fully explained, right middle lobectomy was done. Meticulous histologic examination of the entire tumor revealed well differentiated adenocarcinoma with a mixed acinar and bronchioloalveolar pattern without other components (Fig. 2A). Four cycles of palliative chemotherapy with gemcitabine (1000 mg/m2 , D1 and D8) and carboplatin (area under the curve 6) followed. The pathologic stage was T1bN1M1a and the direct sequencing of the tumor DNA for determining the presence of the
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Fig. 1. A case with acquired resistance to erlotinib and the patient presented with a pancreatic mass. (A) Chest CT showed a 3 cm-sized mass in the right lung of a 50-year-old woman, which was proven to be adenocarcinoma by needle biopsy. (B) Disseminated pleural nodules were found 1 year after surgery and palliative chemotherapy. (C) The multiple nodules on the pleural surface disappeared after treatment with erlotinib. (D) Although a complete remission was maintained in the pleura, and (E) a low attenuated mass was detected in the tail of the pancreas almost 1 year after starting treatment with single agent erlotinib.
EGFR mutation revealed the deletion mutation on exon 19. Multiple pleural nodules and thickening appeared on the chest CT 7 months after chemotherapy (Fig. 1B). Erlotinib was started and this led to near complete response (Fig. 1C). She only complained of a mild skin eruption on her face. However, a low attenuated mass in the tail of pancreas was noted on chest CT almost 1 year after the initiation of single agent erlotinib therapy (Fig. 1E, arrow), although there was no aggravating lesion within her chest (Fig. 1D). Pathologic examination of the biopsied tissue from the pancreatic mass mostly showed areas of spindle-shaped tumor cells with a tiny focus of gland formation (Fig. 2B). Metastasis from lung cancer was the more probable cause of this pancreatic lesion because the immunohistochemical staining for TTF-1 was positive (Fig. 2C) and the same deletion mutation on exon 19 was also found by direct sequencing of the pancreas tumor DNA. Nevertheless, the morphology of the metastatic tumor was quite different from that of the original lung cancer, which showed mixed acinar and bronchioloalveolar features. Spindle-shaped cells with the loss of epithelial polarity, indicating EMT, were noted. Compared with the primary tumor, the metastatic tumor showed the loss of an E-cadherin expression and an increased vimentin expression in immunohistochemistry, which are also indicative of EMT (Fig. 2D). The scorpion ARM test for T790M and FISH for MET amplification were done to exclude the known resistant mechanisms to EGFR-TKIs. There were no secondary mutations or MET amplification (data not shown). This description of the patient was approved by institutional review board (IRB) of Seoul National University Bundang Hospital.
3.2. CL-387,785-resistant subline of HCC827 (designated as HCC827/CLR) showed morphologic and molecular changes of EMT HCC827/CLR cells established by repeated exposure to increasing concentrations of CL-387,785 over a period of 6 months exhibited the resistance more than 100 times to the drug compared with parent cells (Fig. 3A). They showed phenotype changes that are consistent with EMT, such as a spindle-cell morphology and the formation of pseudopodia (Fig. 3B). Accordingly, the expression of epithelial markers, including E-cadherin, -catenin and cytokeratin-8/18, was reduced in the HCC827/CLR cells, whereas the expression of vimentin was increased (Fig. 3C and D). The changes of EMT-related genes expression were also confirmed by cDNA microarray analysis (Table 1). 3.3. The capability of invasion and migration was increased in HCC827/CLR cells Because EMT is associated with increased cellular motility and invasiveness, we further characterized HCC827/CLR cells by Boyden chamber assay for invasion and migration. As we expected, the HCC827/CLR cells showed an enhanced capability for migration (∼1.6-fold) and invasion (∼2.8-fold) (Fig. 4). 3.4. The activity of most of RTKs in HCC827/CLR cells was decreased compared to parent cells We carried out RTK array analysis to find out other bypass signals contributing to the resistance (Fig. 5). The another clone
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Fig. 2. The histopathologic features were evaluated in the primary pulmonary adenocarcinoma and the corresponding pancreatic metastasis. (A) The primary tumor showed well-developed glands with an intra-luminal hobnail appearance. The individual tumor cells had round to oval nuclei, prominent nucleoli with eosinophilic, plump cytoplasm. (B) The metastatic tumor cells revealed sheets of relatively spindle-shaped cells that showed the loss of epithelial polarity and glandular formation. They also had a minimal amount of cytoplasm, they had hyperchromatic nuclei and lacked nucleoli and nuclear molding, which are all features of neuroendocrine carcinoma. (C) A strong nuclear expression of TTF-1 immunostaining was found in the metastatic tumor cells. (D) The loss of E-cadherin and an increased vimentin expression were seen in these metastatic tumors (right panel), as compared with their corresponding primary tumors (left panel).
of HCC827 resistant to gefitinib showed increased p-MET, which was well correlated with the previous report by Engelman et al. However, in HCC827/CLR cells, most of phospho-RTKs including receptor HER family were decreased suggesting that bypass signals might not be the cause of resistance. 3.5. There was no secondary T790M or MET amplification in HCC827/CLR cells Two well-known resistant mechanisms were examined. Because small proportion of T790M which cannot be detectable by direct sequencing would lead to the resistance, we used the more sensitive scorpion ARM method to find the secondary mutation. However, T790M mutation could not be detected and there was no MET amplification by FISH (data not shown). 4. Discussion In this study, we provided more evidences in the clinical and molecular aspects suggesting EMT may contribute to the acquired resistance to EGFR-TKIs. Although the most common cancer metastasizing to pancreas is lung cancer [16], we can very rarely encounter such patients in clinical practice. Because the pancreatic mass in our patient showed very different histology compared to primary lung cancer and there were no other aggravating lesions during erlotinib, we
initially suspected that it would be primarily originated from pancreas. However, we could confirm the metastasis because TTF-1 immunostaining was strong-positive and the same deletion mutation on exon 19 was found in the pancreatic mass. Actually, EGFR tyrosine kinase mutations are very rare in pancreatic cancer [17] and TTF-1 immunostaining is quite specific for thyroid and lung adenocarcinoma [18]. Therefore, it seems to be unreasonable to consider that the primary pancreatic cancer would simultaneously have both of two very rare incidents of EGFR mutation and TTF1 positivity. Although we could not have the exact answer about why the resistant cells having EMT appeared only in pancreas of our patient, it is uncommon to experience cases showing reaggravating lesions outside lung after improvement by EGFR-TKIs while the primary lung cancer remains stable. The environment surrounding the tumor or different drug penetration would affect the selective growth of resistant clone although this phenomenon should be clarified thorough further investigations. Epithelial to mesenchymal transition (EMT) is a process by which cells undergo a morphological change from the epithelial polarized phenotype to the mesenchymal fibroblastoid phenotype. Although EMT leads to enhanced motility and invasion for tumor progression, it can also affect the sensitivity to chemotherapeutic drugs [19,20]. We previously reported A549 lung cancer cells with acquired resistance to gefitinib [15]. For gathering more evidences to support, we have created more resistant sublines to various EGFR-TKIs by using lung cancer cell lines with deletion mutation
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Fig. 3. (A) CL-387,785-resistant cells (HCC827/CLR) were established by long-term exposure to increasing concentrations of CL-387,785. (B) The HCC827 parental cells and the CL-387,785-resistant cells (HCC827/CLR) were assessed for morphologic changes that are consistent with EMT. Compared with the HCC827 cells, the HCC827/CLR cells were observed as spindle-shaped cells (white arrow) with a loss of polarity and increased formation of pseudopodia (black arrow). (C) Loss of E-cadherin and increased expression of vimentin were seen on Western blotting. (D) Immunofluorescence staining was also performed to validate the changes of the marker proteins of either the epithelial or mesenchymal phenotypes. The nuclei were stained with propidium iodide (red fluorescence), and the E-cadherin and vimentin were stained with FITC-conjugated antibodies (green fluorescence). (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)
on exon 19 of EGFR gene. In case of gefitinib-resistant HCC827 cells, p-MET was increased in phospho-RTK assay suggesting MET signal pathway possibly play an important role in resistance to geftinib by trans-activating Akt for survival. This subline is well matched with that of the previous report by Engelman et al. who also used geftinib and HCC827 cells to create the resistance caused by MET amplification [12]. However, among our resistant sublines of HCC827, the Table 1 Microarray analysis for EMT markers. Gene expression level (HCC827/CLR vs. HCC827)a , b
Symbol
Name
2.47 1.91 2.22 1.95 2.68 2.19 2.05 4.25 28.86 −6.67 −7.99 −2.15 −2.33
SNAI2 TWIST1 CDH1 SDC1 COL3A1 SMAD2 SMAD3 MMP1 VIM CDH1 KRT8 KRT18 OCLN
Snail homolog 2 Twist homolog 1 Cadherin 2, type 1, N-cadherin syndecan 1 Collagen, type III, alpha 1 SMAD family member 2 SMAD family member 3 Matrix metallopeptidase 1 Vimentin Cadherin 1, type 1, E-cadherin Keratin 8 Keratin 18 Occludin
a
Fold change. Positive values: higher in HCC827/CLR compared with HCC827 cells; negative values: lower in HCC827/CLR compared with HCC827 cells. b
HCC827/CLR cells were quite different. As we could see in multiple RTK assay, the activity in most of tested RTKs was decreased, even in receptor HER family suggesting that bypass pathway to activate downstream signals would not contribute to the resistance in HCC827/CLR cells. Rather, their morphologic changes prompted us to explore the role of EMT. We could obtain molecular evidences showing EMT in both genetic and protein levels. The enhanced invasion and migration ability of those cells also could be another supporting evidence for EMT. Furthermore, we confirmed that the sensitivity to CL-387,785 was decreased in EMT-induced HCC827 parent cells by TGF-1 treatment as A549/GR cells in our previous study [15] (data not shown). Some studies showed that EMT status has been associated with the sensitivity to EGFR-TKIs in lung cancer cells, xenografts and patients [20–22]. However, the mechanism of EMT-related drug resistance remains unclear. One study suggested that EMT can contribute to EGFR-TKIs resistance by shifting the tumor cells’ predominant reliance on EGFR to other net works, such as those activated by fibroblast growth factor receptor (FGFR), platelet derived growth factor receptor (PDGFR) or ␣51 integrin [23]. Their results were provided by primary lung cancer cells without exposure to EGFR-TKIs and cells with wild-type EGFR. Thus, it needs to evaluate the mechanisms of EMT-induced resistance to EGFRTKIs in the setting of clinical relevance. Our resistant cells may be used to determine mechanisms underlying the EMT process and EMT-induced resistance to EGFR-TKIs.
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Fig. 4. Increased invasion and migration capability in HCC827/CLR cells. The cells were seeded onto either uncoated or Matrigel-coated polycarbonate filters to analyze their migratory and invasive potentials, respectively. The cells were then incubated for 24 h in modified Boyden chambers and the cells that migrated through the filters were stained and counted under a light microscope. *P < 0.005 compared with the HCC827 cells.
At present, it is difficult to figure out the exact incidence of EMTrelated resistance to EGFR-TKIs because re-biopsy to confirm the mechanism of resistance could be performed in only a few cases. However, we expect that more similar case reports and other evidences would be accumulating in near future.
In summary, EMT was found in the tissue sample from our patient and also in a lung cancer cell line with EGFR mutation with acquired resistance to EGFR-TKIs. This suggests that EMT should be considered as one of the possible mechanisms for the acquired resistance to EGFR-TKIs in lung cancer cells. Conflicts of interest All authors have contributed significantly, and they are in agreement with the content of this manuscript. The authors state that this manuscript have no conflicts of interest to disclose. Acknowledgements This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A084578) (to JHC). References
Fig. 5. Phospho-receptor tyrosine kinase array analysis. Cells were grown to confluence followed by protein extraction. Cell lysates were incubated in each membrane containing antibodies to 42 different RTKs. The membranes were washed and incubated with a pan-antiphosphotyrosine antibody to measure the levels of active receptor. The membranes were exposed to film.
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[5] Uramoto H, Mitsudomi T. Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer? Br J Cancer 2007;96:857–63. [6] Sequist LV, Bell DW, Lynch TJ, Haber DA. Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer. J Clin Oncol 2007;25:587–95. [7] Jeon SY, Rho JK, Choi YJ, Koh JS, Lee JK, Kim CH, et al. What’s your treatment option in a patient with impending respiratory failure due to disseminated lung cancer? Resp Med CME 2008;1:96–9. [8] Perez-Soler R, Chachoua A, Hammond LA, Rowinsky EK, Huberman M, Karp D, et al. Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer. J Clin Oncol 2004;22:3238–47. [9] Yun CH, Menqwasser KE, Toms AV, Woo MS, Greulich H, Wong KK, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci U S A 2008;105:2070–5. [10] Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005;2:e73. [11] Kosaka T, Yatabe Y, Endoh H, Yoshida K, Hida T, Tsuboi M, et al. Analysis of epidermal growth factor receptor gene mutation in patients with nonsmall cell lung cancer and acquired resistance to gefitinib. Clin Cancer Res 2006;12:5764–9. [12] Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 2007;316:1039–43. [13] Guo A, Villen J, Kornhauser J, Lee KA, Stokes MP, Rikova K, et al. Signaling networks assembled by oncogenic EGFR and c-met. Proc Natl Acad Sci U S A 2008;105:692–7. [14] Guix M, Faber AC, Wang SE, Olivares MG, Song Y, Qu S, et al. Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGFbinding proteins. J Clin Invest 2008;118:2609–19.
[15] Rho JK, Choi YJ, Lee JK, Ryoo BY, Na II, Yang SH, et al. Epithelial to mesenchymal transition derived from repeated exposure to gefitinib determines the sensitivity to EGFR inhibitors in A549, a non-small cell lung cancer cell line. Lung Cancer 2009;63:219–26. [16] Sato M, Okumura T, Kaito K, Kiyoshima M, Asato Y, Uchiumi K, et al. Usefulness of FDG-PET/CT in the detection of pancreatic metastases from lung cancer. Ann Nucl Med 2009;23:49–57. [17] Lee J, Jang KT, Ki CS, Lim T, Park YS, Lim HY, et al. Impact of epidermal growth factor receptor (EGFR) kinase mutations, EGFR gene amplifications, and KRAS mutations on survival of pancreatic adenocarcinoma. Cancer 2007;109:1561–9. [18] Moldvay J, Jackel M, Bogos K, Soltész I, Agócs L, Kovács G, et al. The role of TTF-1 in differentiating primary and metastatic lung adenocarcinomas. Pathol Oncol Res 2004;10:85–8. [19] Shah AN, Gallick GE. Src, chemoresistance and epithelial to mesenchymal transition: are they related? Anticancer Drugs 2007;18:371–5. [20] Thomson S, Buck E, Petti F, Griffin G, Brown E, Ramnarine N, et al. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res 2005;65:9455–62. [21] Witta SE, Gemmill RM, Hirsch FR, Coldren CD, Hedman K, Ravdel L, et al. Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines. Cancer Res 2006;66:944–50. [22] Yauch RL, Januario T, Eberhard DA, Cavet G, Zhu W, Fu L, et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 2005;11:8686–98. [23] Thomson S, Petti F, Sujka-Kwok I, Epstein D, Haley JD. Kinase switching in mewnchymal-like non-small cell lung cancer lines contributes to EGFR inhibitor resistance through pathway redundancy. Clin Exp Metast 2008;25:843–54.
Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs Jin-Haeng Chung a,1 , Jin Kyung Rho b,1 , Xianhua Xu a , Jong Seok Lee c , Ho Il Yoon c , Choon Taek Lee c , Yun Jung Choi b , Hye-Ryoun Kim b , Cheol Hyeon Kim b , Jae Cheol Lee b,∗ a
Department of Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Gyeonggi-do, Republic of Korea Department of Internal Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological & Medical Science, 215-4, Gongneung-dong, Nowon-gu, Seoul 139-706, Republic of Korea c Department of Internal Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Gyeonggi-do, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 26 May 2010 Received in revised form 22 November 2010 Accepted 23 November 2010 Keywords: Epithelial to mesenchymal transition EGFR Resistance Lung cancer
a b s t r a c t Background: Epithelial-to-mesenchymal transition (EMT), which was related with an acquired resistance to gefitinib, was found in the A549 lung cancer cell line. However, the clinical feasibility of this finding is still questionable. Here, we investigated whether EMT could be detected in a more clinically suitable situation using patient’s tumor and cells with deletion mutation on exon 19 of EGFR gene. Methods: HCC827 cell line was used to establish the subline resistant to EGFR-TKIs. The induction of EMT was analyzed by immunostainings and Western blots in resistant cells and biopsied tissue from a patient with acquired resistance to erlotinib. Migration and invasion assay was performed to characterize the resistant cells. EMT-related genes expression was evaluated by cDNA microarray. Phospho-receptor tyrosine kinase array analysis was carried out to find bypass activating signals such as MET. Results: We found that EMT developed in a lung cancer patient who had an acquired resistance to erlotinib while there were no known resistant mechanisms such as T790M and MET amplification. CL-387,785resistant cells (HCC827/CLR) were obtained by long-term exposure to increasing concentrations of CL387,785 (an irreversible EGFR-TKI). The morphological and molecular maker changes compatible with EMT were also found in HCC827/CLR cells. However, there were also no secondary T790M mutation and MET amplification. Furthermore, the activity of most of tested RTKs including receptor HER family was decreased suggesting that there was no bypass activating signal leading to resistance. These cells showed an enhanced capability for migration (∼1.6-fold) and invasion (∼2.8-fold). Conclusion: EMT should be considered as one of possible mechanisms for the acquired resistance to EGFR-TKIs in lung cancer cells. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction EGFR-TKIs such as gefitinib and erlotinib have been used for the treatment of non-small cell lung cancer and they have shown minimal toxicity and a modest effect [1,2]. Several clinical and molecular factors have been determined to be related with a better response and survival with using these novel drugs [3–5]. Among them, EGFR mutations, including deletion mutations on exon 19 and point mutations on exon 21, have been suggested to be the most reliable predictors [5,6]. Sometimes EGFR-TKIs have shown a rapid and dramatic response for allowing a patient to recover from the impending respiratory failure caused by disseminated lung cancer
∗ Corresponding author. Tel.: +82 2 9701206. E-mail address: [email protected] (J.C. Lee). 1 Both authors contributed equally to this study. 0169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2010.11.011
[7]. However, drug resistance eventually develops in most patients despite the initial good response, and this has limited the median time to progression to almost 1 year [8]. Two major resistance mechanisms have currently been revealed. T790M secondary mutation increases the affinity of the oncogenic mutant EGFR for ATP, and this leads to the reduced efficacy of EGFR-TKIs [9]. Almost half of the patients with acquired resistance seem to have this mutation [10,11]. MET amplification is another mechanism to escape the anti-tumor effect of EGFR-TK inhibitors, which allows cell survival by persistent Akt signaling via bypassing MET signaling when the EGFR signal is blocked in the presence of EGFR-TKI. MET amplification has been found in around 20% of the patients with acquired resistance [12,13]. Through considerable efforts have been made to clarify unknown mechanisms that induce resistance, some other possible suggestions have been presented. Guix et al. demonstrated that activation of insulin-like growth factor receptor (IGFR) signaling
J.-H. Chung et al. / Lung Cancer 73 (2011) 176–182
and the loss of IGF binding proteins (IGFBPs) were involved in the adaptive change of tumor cells for acquiring resistance to gefitinib, although this was not confirmed in any lung cancer cell lines [14]. In addition, we previously reported that epithelial-to-mesenchymal transition (EMT) would contribute to the decreased efficacy of therapy due to the resistance to gefitinib [15]. However, the clinical feasibility of EMT as a mechanism of resistance remains unclear because it was only found in one cell line, A549, which has a K-ras mutation, and this indicates that this cell line is not an appropriate model for acquired resistance to EGFR-TKIs. Therefore in this study, we provide more evidences showing that EMT may contribute the development of resistance during longterm exposure to EGFR-TKIs. 2. Materials and methods 2.1. Cell cultures and reagents HCC827 cells with deletion mutation on exon 19 of EGFR gene were cultured in RPMI 1640 (Invitrogen, Carsbad, CA) that contained 10% fetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin (Invitrogen) at 37 ◦ C in a 5% CO2 atmosphere. CL-387,785 (an irreversible EGFR-TKI) was purchased from Calbiochem (San Diego, CA). 2.2. Establishment of the gefitinib/CL-387,785-resistant cell lines The HCC827/CLR cells were established by using a previously described method [15]. Over a period of 6 months, the HCC827 cells in culture were continuously exposed to increasing concentrations of CL-387,785. The surviving cells were cloned and the CL-387,785resistant cell line was designated as HCC827/CLR. These cell lines can survive exposure to over 100 nM CL-387,785.
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phosphate-buffered saline (PBS) and processed further with using a DAKO EnVision kit (DAKO, Los Angles, CA), as directed by the manufacturer. The color was developed with 3,3 -diaminobenzindine (DAB) that contained 0.3% H2 O2 . Primary antibodies against the following antigens were used: E-cadherin, vimentin and thyroid transcripton factor-1 (TTF-1). 2.7. Fluorescent immunocytochemistry To identify the induction of EMT, immunocytochemical evaluation of E-cadherin and vimentin was performed by using a previously described method [15]. 2.8. cDNA microarray Total RNA was isolated from HCC827 and HCC827/CLR cells using TRIZOL reagent (Invitrogen, Carlsbad, CA), followed by RNeasy Mini Kit (Qiagen, Valencia, CA). For control and test RNAs, the synthesis of target cRNA probes and hybridization were performed using Agilent’s Low RNA Input Linear Amplification kit (Agilent Technology, USA) according to the manufacturer’s instructions. Labeled sample (Cy5 CTP) and reference (Cy3 CTP) were mixed and co-hybridized overnight on the same 44K Agilent’s Human Oligo Microarray. The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technology, Palo Alto, CA). All data normalization and selection of fold-changed genes were performed using GeneSpringGX 7.3 (Agilent Technology). The averages of normalized ratios were calculated by dividing the average of normalized signal channel intensity by the average of normalized control channel intensity.
2.3. MTT assay
2.9. Phospho-receptor tyrosine kinase (RTK) array analysis
Briefly, the cells were seeded onto 96-well plates overnight. Each of the drugs was added in a dose dependent manner and the cells were incubated for 72 h. The viability of the cells was determined by using the MTT assay.
It was performed according to manufacturer’s instructions (R&D Systems, Minneapolis, MN). Briefly, 500 g of lysates was incubated in each membrane. The arrays were washed and incubated with a horseradish peroxidase-conjugated phosphor-tyrosine detection antibody, treated with ECL and exposed to film. Intensity of each spot detected with a phospho-RTK array was measured by ImageQuant software (GE healthcare).
2.4. Immunoblot analysis The membrane was probed with antibody against E-cadherin (DakoCytomation, Carpinteria, CA), -catenin (Cell signaling Technology, Beverly, MA), cytokeratin-8/18 (Santa Cruz Biotechnology, Santa Cruz, CA), vimentin (Calbiochem, San Diego, CA) and -actin (Cell signaling) as the first antibody, and then the membrane was treated with horseradish-peroxidase-conjugated secondary antibody. The membrane was developed using an ECL kit (Amersham Biosciences, Piscataway, NJ). 2.5. Migration and invasion assays The cell migration and invasion assays were done according to a previously described method [15]. The results were expressed as means ± SDs. 2.6. Immunohistochemistry For immunohistochemical analysis, the paraffin sections (4 m thick) were deparaffinized with xylene, rinsed thoroughly with ethanol and then soaked in 0.03% hydrogen peroxide in methanol to inactivate the endogenous peroxidase activity. The sections were incubated with either 10% goat serum or 10% rabbit serum, and then they were covered with the primary antibodies, washed with
3. Results 3.1. EMT was found in a patient with acquired resistance to erlotinib A 50-year-old woman was admitted to Seoul National University Bundang Hospital in August 2006 for the evaluation of a lung mass. Chest CT showed an about 3 cm-sized mass on the right middle lobe of the lung without enlargement of the mediastinal lymph nodes (Fig. 1A). The acinar type of adenocarcinoma was diagnosed by percutaneous fine needle aspiration biopsy. However, disseminated tiny nodules were found on the pleura during the operation, and this indicated the advanced stage of her disease. Because the family of our patient strongly wanted to remove the mass although the status of her disease was fully explained, right middle lobectomy was done. Meticulous histologic examination of the entire tumor revealed well differentiated adenocarcinoma with a mixed acinar and bronchioloalveolar pattern without other components (Fig. 2A). Four cycles of palliative chemotherapy with gemcitabine (1000 mg/m2 , D1 and D8) and carboplatin (area under the curve 6) followed. The pathologic stage was T1bN1M1a and the direct sequencing of the tumor DNA for determining the presence of the
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Fig. 1. A case with acquired resistance to erlotinib and the patient presented with a pancreatic mass. (A) Chest CT showed a 3 cm-sized mass in the right lung of a 50-year-old woman, which was proven to be adenocarcinoma by needle biopsy. (B) Disseminated pleural nodules were found 1 year after surgery and palliative chemotherapy. (C) The multiple nodules on the pleural surface disappeared after treatment with erlotinib. (D) Although a complete remission was maintained in the pleura, and (E) a low attenuated mass was detected in the tail of the pancreas almost 1 year after starting treatment with single agent erlotinib.
EGFR mutation revealed the deletion mutation on exon 19. Multiple pleural nodules and thickening appeared on the chest CT 7 months after chemotherapy (Fig. 1B). Erlotinib was started and this led to near complete response (Fig. 1C). She only complained of a mild skin eruption on her face. However, a low attenuated mass in the tail of pancreas was noted on chest CT almost 1 year after the initiation of single agent erlotinib therapy (Fig. 1E, arrow), although there was no aggravating lesion within her chest (Fig. 1D). Pathologic examination of the biopsied tissue from the pancreatic mass mostly showed areas of spindle-shaped tumor cells with a tiny focus of gland formation (Fig. 2B). Metastasis from lung cancer was the more probable cause of this pancreatic lesion because the immunohistochemical staining for TTF-1 was positive (Fig. 2C) and the same deletion mutation on exon 19 was also found by direct sequencing of the pancreas tumor DNA. Nevertheless, the morphology of the metastatic tumor was quite different from that of the original lung cancer, which showed mixed acinar and bronchioloalveolar features. Spindle-shaped cells with the loss of epithelial polarity, indicating EMT, were noted. Compared with the primary tumor, the metastatic tumor showed the loss of an E-cadherin expression and an increased vimentin expression in immunohistochemistry, which are also indicative of EMT (Fig. 2D). The scorpion ARM test for T790M and FISH for MET amplification were done to exclude the known resistant mechanisms to EGFR-TKIs. There were no secondary mutations or MET amplification (data not shown). This description of the patient was approved by institutional review board (IRB) of Seoul National University Bundang Hospital.
3.2. CL-387,785-resistant subline of HCC827 (designated as HCC827/CLR) showed morphologic and molecular changes of EMT HCC827/CLR cells established by repeated exposure to increasing concentrations of CL-387,785 over a period of 6 months exhibited the resistance more than 100 times to the drug compared with parent cells (Fig. 3A). They showed phenotype changes that are consistent with EMT, such as a spindle-cell morphology and the formation of pseudopodia (Fig. 3B). Accordingly, the expression of epithelial markers, including E-cadherin, -catenin and cytokeratin-8/18, was reduced in the HCC827/CLR cells, whereas the expression of vimentin was increased (Fig. 3C and D). The changes of EMT-related genes expression were also confirmed by cDNA microarray analysis (Table 1). 3.3. The capability of invasion and migration was increased in HCC827/CLR cells Because EMT is associated with increased cellular motility and invasiveness, we further characterized HCC827/CLR cells by Boyden chamber assay for invasion and migration. As we expected, the HCC827/CLR cells showed an enhanced capability for migration (∼1.6-fold) and invasion (∼2.8-fold) (Fig. 4). 3.4. The activity of most of RTKs in HCC827/CLR cells was decreased compared to parent cells We carried out RTK array analysis to find out other bypass signals contributing to the resistance (Fig. 5). The another clone
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Fig. 2. The histopathologic features were evaluated in the primary pulmonary adenocarcinoma and the corresponding pancreatic metastasis. (A) The primary tumor showed well-developed glands with an intra-luminal hobnail appearance. The individual tumor cells had round to oval nuclei, prominent nucleoli with eosinophilic, plump cytoplasm. (B) The metastatic tumor cells revealed sheets of relatively spindle-shaped cells that showed the loss of epithelial polarity and glandular formation. They also had a minimal amount of cytoplasm, they had hyperchromatic nuclei and lacked nucleoli and nuclear molding, which are all features of neuroendocrine carcinoma. (C) A strong nuclear expression of TTF-1 immunostaining was found in the metastatic tumor cells. (D) The loss of E-cadherin and an increased vimentin expression were seen in these metastatic tumors (right panel), as compared with their corresponding primary tumors (left panel).
of HCC827 resistant to gefitinib showed increased p-MET, which was well correlated with the previous report by Engelman et al. However, in HCC827/CLR cells, most of phospho-RTKs including receptor HER family were decreased suggesting that bypass signals might not be the cause of resistance. 3.5. There was no secondary T790M or MET amplification in HCC827/CLR cells Two well-known resistant mechanisms were examined. Because small proportion of T790M which cannot be detectable by direct sequencing would lead to the resistance, we used the more sensitive scorpion ARM method to find the secondary mutation. However, T790M mutation could not be detected and there was no MET amplification by FISH (data not shown). 4. Discussion In this study, we provided more evidences in the clinical and molecular aspects suggesting EMT may contribute to the acquired resistance to EGFR-TKIs. Although the most common cancer metastasizing to pancreas is lung cancer [16], we can very rarely encounter such patients in clinical practice. Because the pancreatic mass in our patient showed very different histology compared to primary lung cancer and there were no other aggravating lesions during erlotinib, we
initially suspected that it would be primarily originated from pancreas. However, we could confirm the metastasis because TTF-1 immunostaining was strong-positive and the same deletion mutation on exon 19 was found in the pancreatic mass. Actually, EGFR tyrosine kinase mutations are very rare in pancreatic cancer [17] and TTF-1 immunostaining is quite specific for thyroid and lung adenocarcinoma [18]. Therefore, it seems to be unreasonable to consider that the primary pancreatic cancer would simultaneously have both of two very rare incidents of EGFR mutation and TTF1 positivity. Although we could not have the exact answer about why the resistant cells having EMT appeared only in pancreas of our patient, it is uncommon to experience cases showing reaggravating lesions outside lung after improvement by EGFR-TKIs while the primary lung cancer remains stable. The environment surrounding the tumor or different drug penetration would affect the selective growth of resistant clone although this phenomenon should be clarified thorough further investigations. Epithelial to mesenchymal transition (EMT) is a process by which cells undergo a morphological change from the epithelial polarized phenotype to the mesenchymal fibroblastoid phenotype. Although EMT leads to enhanced motility and invasion for tumor progression, it can also affect the sensitivity to chemotherapeutic drugs [19,20]. We previously reported A549 lung cancer cells with acquired resistance to gefitinib [15]. For gathering more evidences to support, we have created more resistant sublines to various EGFR-TKIs by using lung cancer cell lines with deletion mutation
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Fig. 3. (A) CL-387,785-resistant cells (HCC827/CLR) were established by long-term exposure to increasing concentrations of CL-387,785. (B) The HCC827 parental cells and the CL-387,785-resistant cells (HCC827/CLR) were assessed for morphologic changes that are consistent with EMT. Compared with the HCC827 cells, the HCC827/CLR cells were observed as spindle-shaped cells (white arrow) with a loss of polarity and increased formation of pseudopodia (black arrow). (C) Loss of E-cadherin and increased expression of vimentin were seen on Western blotting. (D) Immunofluorescence staining was also performed to validate the changes of the marker proteins of either the epithelial or mesenchymal phenotypes. The nuclei were stained with propidium iodide (red fluorescence), and the E-cadherin and vimentin were stained with FITC-conjugated antibodies (green fluorescence). (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)
on exon 19 of EGFR gene. In case of gefitinib-resistant HCC827 cells, p-MET was increased in phospho-RTK assay suggesting MET signal pathway possibly play an important role in resistance to geftinib by trans-activating Akt for survival. This subline is well matched with that of the previous report by Engelman et al. who also used geftinib and HCC827 cells to create the resistance caused by MET amplification [12]. However, among our resistant sublines of HCC827, the Table 1 Microarray analysis for EMT markers. Gene expression level (HCC827/CLR vs. HCC827)a , b
Symbol
Name
2.47 1.91 2.22 1.95 2.68 2.19 2.05 4.25 28.86 −6.67 −7.99 −2.15 −2.33
SNAI2 TWIST1 CDH1 SDC1 COL3A1 SMAD2 SMAD3 MMP1 VIM CDH1 KRT8 KRT18 OCLN
Snail homolog 2 Twist homolog 1 Cadherin 2, type 1, N-cadherin syndecan 1 Collagen, type III, alpha 1 SMAD family member 2 SMAD family member 3 Matrix metallopeptidase 1 Vimentin Cadherin 1, type 1, E-cadherin Keratin 8 Keratin 18 Occludin
a
Fold change. Positive values: higher in HCC827/CLR compared with HCC827 cells; negative values: lower in HCC827/CLR compared with HCC827 cells. b
HCC827/CLR cells were quite different. As we could see in multiple RTK assay, the activity in most of tested RTKs was decreased, even in receptor HER family suggesting that bypass pathway to activate downstream signals would not contribute to the resistance in HCC827/CLR cells. Rather, their morphologic changes prompted us to explore the role of EMT. We could obtain molecular evidences showing EMT in both genetic and protein levels. The enhanced invasion and migration ability of those cells also could be another supporting evidence for EMT. Furthermore, we confirmed that the sensitivity to CL-387,785 was decreased in EMT-induced HCC827 parent cells by TGF-1 treatment as A549/GR cells in our previous study [15] (data not shown). Some studies showed that EMT status has been associated with the sensitivity to EGFR-TKIs in lung cancer cells, xenografts and patients [20–22]. However, the mechanism of EMT-related drug resistance remains unclear. One study suggested that EMT can contribute to EGFR-TKIs resistance by shifting the tumor cells’ predominant reliance on EGFR to other net works, such as those activated by fibroblast growth factor receptor (FGFR), platelet derived growth factor receptor (PDGFR) or ␣51 integrin [23]. Their results were provided by primary lung cancer cells without exposure to EGFR-TKIs and cells with wild-type EGFR. Thus, it needs to evaluate the mechanisms of EMT-induced resistance to EGFRTKIs in the setting of clinical relevance. Our resistant cells may be used to determine mechanisms underlying the EMT process and EMT-induced resistance to EGFR-TKIs.
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Fig. 4. Increased invasion and migration capability in HCC827/CLR cells. The cells were seeded onto either uncoated or Matrigel-coated polycarbonate filters to analyze their migratory and invasive potentials, respectively. The cells were then incubated for 24 h in modified Boyden chambers and the cells that migrated through the filters were stained and counted under a light microscope. *P < 0.005 compared with the HCC827 cells.
At present, it is difficult to figure out the exact incidence of EMTrelated resistance to EGFR-TKIs because re-biopsy to confirm the mechanism of resistance could be performed in only a few cases. However, we expect that more similar case reports and other evidences would be accumulating in near future.
In summary, EMT was found in the tissue sample from our patient and also in a lung cancer cell line with EGFR mutation with acquired resistance to EGFR-TKIs. This suggests that EMT should be considered as one of the possible mechanisms for the acquired resistance to EGFR-TKIs in lung cancer cells. Conflicts of interest All authors have contributed significantly, and they are in agreement with the content of this manuscript. The authors state that this manuscript have no conflicts of interest to disclose. Acknowledgements This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A084578) (to JHC). References
Fig. 5. Phospho-receptor tyrosine kinase array analysis. Cells were grown to confluence followed by protein extraction. Cell lysates were incubated in each membrane containing antibodies to 42 different RTKs. The membranes were washed and incubated with a pan-antiphosphotyrosine antibody to measure the levels of active receptor. The membranes were exposed to film.
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