Properties of resistant cells generated from lung cancer cell lines treated with EGFR inhibitors

BMC Cancer

Gargi Ghosh²  Xiaojun Lian³  Stephen J Kron¹  Sean P Palecek³ 

[1] Ludwig Center for Metastasis Research, University of Chicago, Chicago, IL 60637, USA

[2] Department of Mechanical Engineering, University of Michigan, Dearborn, MI 48128, USA

[3] Department of Chemical and Biological Engineering, University of Wisconsin, Madison, 1415 Engineering Drive, Madison, WI 53706, USA

关键词: Side population;    Tumor spheroids;    Cancer stem cells;    Erlotinib;    EGFR tyrosine kinase;   

Others  :  1080512
DOI  :  10.1186/1471-2407-12-95

 received in 2011-08-03, accepted in 2012-03-20,  发布年份 2012

【 摘 要 】

Background

Epidermal growth factor receptor (EGFR) signaling plays an important role in non-small cell lung cancer (NSCLC) and therapeutics targeted against EGFR have been effective in treating a subset of patients bearing somatic EFGR mutations. However, the cancer eventually progresses during treatment with EGFR inhibitors, even in the patients who respond to these drugs initially. Recent studies have identified that the acquisition of resistance in approximately 50% of cases is due to generation of a secondary mutation (T790M) in the EGFR kinase domain. In about 20% of the cases, resistance is associated with the amplification of MET kinase. In the remaining 30-40% of the cases, the mechanism underpinning the therapeutic resistance is unknown.

Methods

An erlotinib resistant subline (H1650-ER1) was generated upon continuous exposure of NSCLC cell line NCI-H1650 to erlotinib. Cancer stem cell like traits including expression of stem cell markers, enhanced ability to self-renew and differentiate, and increased tumorigenicity in vitro were assessed in erlotinib resistant H1650-ER1 cells.

Results

The erlotinib resistant subline contained a population of cells with properties similar to cancer stem cells. These cells were found to be less sensitive towards erlotinib treatment as measured by cell proliferation and generation of tumor spheres in the presence of erlotinib.

Conclusions

Our findings suggest that in cases of NSCLC accompanied by mutant EGFR, treatment targeting inhibition of EGFR kinase activity in differentiated cancer cells may generate a population of cancer cells with stem cell properties.

【 授权许可】

   
2012 Ghosh et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Sebolt-Leopold JS, English JM: Mechanisms of drug inhibition of signalling molecules. Nature 2006, 441:457-62.
• [2]Laskin JJ, Sandler AB: Epidermal growth factor receptor: a promising target in solid tumours. Cancer Treat Rev 2004, 30:1-17.
• [3]Madhusudan S, Ganesan TS: Tyrosine kinase inhibitors in cancer therapy. Clin Biochem 2004, 37:618-635.
• [4]Yarden Y: The EGFR family and its ligands in human cancer: signaling mechanisms and therapeutic opportunities. Eur J Cancer 2001, 37:S3-S8.
• [5]Vlahovic G, Crawford J: Activation of tyrosine kinases in cancer. The Oncologist 2003, 8:531-538.
• [6]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.
• [7]Shepherd FA, Pereira JR, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S, et al.: Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005, 353:123-132.
• [8]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, 25:587-595.
• [9]Rossell R, Moran T, Queralt C, Porta R, Cardenal F, Majem M, et al.: Screening for Epidermal Growth Factor Receptor mutations in lung Cancer. N Engl J Med 2009, 361:958-967.
• [10]Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson M, et al.: EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 2005, 352:786-792.
• [11]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.
• [12]Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L, et al.: MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci USA 2007, 104:20932-20937.
• [13]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, 316:1039-1043.
• [14]Kwak EL, Sordella R, Bell DW, Godin-Heymann N, Okimoto RA, Brannigan BW, et al.: Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc Natl Acad Sci USA 2005, 102:7665-7670.
• [15]Yao Z, Fenoglio S, Gao DC, Camiolo M, Stiles B, Lindsted T, et al.: TGF-beta IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer. Proc Natl Acad Sci USA 2010, 107:15535-15540.
• [16]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, 65:9455-9462.
• [17]Thomson S, Petti F, Sujka-Kwok I, Epstein D, Haley JD: Kinase switching in mesenchymal-like non-small cell lung cancer lines contributes to EGFR inhibitor resistance through pathway redundancy. Clin Exp Metastasis 2008, 25:843-854.
• [18]Uramoto H, Iwata T, Onitsuka T, Shimokawa H, Hanagiri T, Oyama T: Epiethelial-mesenchymal transition in EGFR-TKI acquired resistant lung adenocarcinoma. Anticancer Res 2010, 30:2513-2518.
• [19]Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al.: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008, 133:704-715.
• [20]Bonnet D, Dick JE: Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997, 3:730-737.
• [21]Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003, 100:3983-3988.
• [22]Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al.: Identification of human brain tumour initiating cells. Nature 2004, 432:396-401.
• [23]Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L, et al.: Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci USA 2009, 106:16281-16286.
• [24]Eramo A, Haas TL, De Maria R: Lung cancer stem cells: tools and targets to fight lung cancer. Oncogene 2010, 29:4625-4635.
• [25]Gu G, Yuan J, Wills M, Kasper S: Prostate cancer cells with stem cell characteristics reconstitute the original human tumor in vivo. Cancer Res 2007, 67:4807-4815.
• [26]Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, et al.: Identification of pancreatic cancer stem cells. Cancer Res 2007, 67:1030-1037.
• [27]Sullivan JP, Minna JD, Shay JW: Evidence for self-renewing lung cancer stem cells and their implications in tumor initiation, progression, and targeted therapy. Cancer Metastasis Rev 2010, 29:61-72.
• [28]Wicha MS, Liu S, Dontu G: Cancer stem cells: an old idea--a paradigm shift. Cancer Res 2006, 66:1883-1890. discussion 95-6
• [29]Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC: Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996, 183:1797-1806.
• [30]Telford WG, Bradford J, Godfrey W, Robey RW, Bates SE: Side population analysis using a violet-excited cell-permeable DNA binding dye. Stem Cells 2007, 25:1029-1036.
• [31]Ghosh G, Yan X, Lee AG, Kron SJ, Palecek SP: Quantifying the sensitivities of EGF receptor (EGFR) tyrosine kinase inhibitors in drug resistant non-small cell lung cancer (NSCLC) cells using hydrogel-based peptide array. Biosens Bioelectron 2010, 26:424-431.
• [32]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, 11:8686-8698.
• [33]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, 63:219-226.
• [34]Beier D, Hau P, Proescholdt M, Lohmeier A, Wischhusen J, Oefner PJ, et al.: CD133(+) and CD133(-) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res 2007, 67:4010-4015.
• [35]Eramo A, Lotti F, Sette G, Pillozi E, Biffoni M, Virgillo AD, et al.: Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death and Differentiation 2008, 15:504-514.
• [36]Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, et al.: Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007, 1:313-323.
• [37]Miki J, Furusato B, Li H, Gu Y, Takahashi H, Egawa S, et al.: Identification of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized primary nonmalignant and malignant tumor-derived human prostate epithelial cell lines and in prostate cancer specimens. Cancer Res 2007, 67:3153-3161.
• [38]Lengner CJ, Welstead GG, Jaenisch R: The pluripotency of regulator OCT4: A role in somatic cells? Cell Cycle 2008, 7:725-728.
• [39]Chiou SH, Wang ML, Chou YT, Chen CJ, Hong CF, Hseih WJ, et al.: Coexpression of OCT4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell like properties and epithelial mesenchymal transdifferentiation. Cancer Res 2010, 70:10433-10444.
• [40]Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, et al.: Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 2003, 423:302-305.
• [41]Niwa H, Burdon T, Chambers I, Smith A: Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998, 12:2048-2060.
• [42]Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, et al.: Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003, 113:643-655.
• [43]Colburn NH, Bruegge WF, Bates JR, Gray RH, Rossen JD, Kelsey WH, et al.: Correlation of anchorage-independent growth with tumorigenicity of chemically transformed mouse epidermal cells. Cancer Res 1978, 38:624-634.
• [44]Hwang-Verslues WW, Kuo WH, Chang PH, Pan CC, Wang HH, Tsai ST, et al.: Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers. PLoS One 2009, 4:e8377.
• [45]Benavente S, Huang S, Armstrong EA, Chi A, Hsu KT, Wheeler DL, et al.: Establishment and characterization of a model of acquired resistance to epidermal growth factor receptor targeting agents in human cancer cells. Clin Cancer Res 2009, 15:1585-1592.