Resistance of oral squamous cell carcinoma cells to cetuximab is associated with EGFR insensitivity and enhanced stem cell-like potency

  • Authors:
    • Yuichi Ohnishi
    • Yuki Minamino
    • Kenji  Kakudo
    • Masami Nozaki
  • View Affiliations

  • Published online on: June 12, 2014     https://doi.org/10.3892/or.2014.3258
  • Pages: 780-786
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Abstract

Cetuximab, a specific anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is used in cancer treatment. Although development of resistance to cetuximab is well recognized, the underlying mechanisms remain unclear. In the present study, we characterized cetuximab-resistant oral squamous cell carcinoma (OSCC) cell lines. The human OSCC cell lines HSC3, HSC4 and SAS were used in the present study. Effects of inhibitors including cetuximab on growth in cells were assessed by MTT assays. Southern blotting and immunofluorescence analysis were performed to examine protein expression and localization. Sphere formation was used to characterize stem cell-like properties. Floating aggregation culture was used for anchorage-independent growth. Cetuximab inhibited proliferation of HSC3 and HSC4 cells, but not SAS cells. Proliferation of all three cell lines was inhibited by the EGFR/ErbB2/ErbB4 inhibitor II. The EGFR inhibitor AG1478 strongly inhibited HSC3 and HSC4 proliferation, but that of SAS cells only moderately. EGFR proteins were localized on cell surface and phosphorylated in all three cell lines. SAS cells could proliferate in serum-free monolayer culture and formed spheres from single cells in floating culture. HSC3 and HSC4 could not proliferate under serum-free culture conditions and could not form spheres. Growth of SAS spheres required serum, and was inhibited by both AG1478 and cetuximab. Thus, cetuximab-resistant SAS cells not only engaged in EGFR-independent growth but also exhibited stem cell-like properties. However, growth was EGFR-dependent in aggregation culture, and the SAS cell aggregates became cetuximab-sensitive. This suggests that cetuximab sensitivity is not only cell-type-dependent but is also affected by the growth microenvironment.

References

1 

Yarden Y and Sliwkowski MX: Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2:127–137. 2001. View Article : Google Scholar : PubMed/NCBI

2 

Harris RC, Chung E and Coffey RJ: EGF receptor ligands. Exp Cell Res. 284:2–13. 2003. View Article : Google Scholar

3 

Arteaga CL and Baselga J: Tyrosine kinase inhibitors: why does the current process of clinical development not apply to them? Cancer Cell. 5:525–531. 2004. View Article : Google Scholar : PubMed/NCBI

4 

Galer CE, Corey CL, Wang Z, Younes MN, Gomez-Rivera F, Jasser SA, Ludwig DL, El-Naggar AK, Weber RS and Myers JN: Dual inhibition of epidermal growth factor receptor and insulin-like growth factor receptor I: reduction of angiogenesis and tumor growth in cutaneous squamous cell carcinoma. Head Neck. 33:189–198. 2011. View Article : Google Scholar : PubMed/NCBI

5 

Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, Lindeman N, Gale CM, Zhao X, Christensen J, Kosaka T, Holmes AJ, Rogers AM, Cappuzzo F, Mok T, Lee C, Johnson BE, Cantley LC and Jänne PA: MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 316:1039–1043. 2007. View Article : Google Scholar

6 

Ang KK, Berkey BA, Tu X, Zhang HZ, Katz R, Hammond EH, Fu KK and Milas L: Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res. 62:7350–7356. 2002.PubMed/NCBI

7 

Chung CH, Zhang Q, Hammond EM, Trotti AM III, Wang H, Spencer S, Zhang HZ, Cooper J, Jordan R, Rotman MH and Ang KK: Integrating epidermal growth factor receptor assay with clinical parameters improves risk classification for relapse and survival in head-and-neck squamous cell carcinoma. Int J Radiat Oncol Biol Phys. 81:331–338. 2011. View Article : Google Scholar : PubMed/NCBI

8 

Galizia G, Lieto E, De Vita F, Orditura M, Castellano P, Troiani T, Imperatore V and Ciardiello F: Cetuximab, a chimeric human mouse anti-epidermal growth factor receptor monoclonal antibody, in the treatment of human colorectal cancer. Oncogene. 26:3654–3660. 2007. View Article : Google Scholar : PubMed/NCBI

9 

Li S, Schmitz KR, Jeffrey PD, Wiltzius JJ, Kussie P and Ferguson KM: Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell. 7:301–311. 2005. View Article : Google Scholar : PubMed/NCBI

10 

López-Albaitero A and Ferris RL: Immune activation by epidermal growth factor receptor-specific monoclonal antibody therapy for head and neck cancer. Arch Otolaryngol Head Neck Surg. 133:1277–1281. 2007.PubMed/NCBI

11 

Kurai J, Chikumi H, Hashimoto K, Yamaguchi K, Yamasaki A, Sako T, Touge H, Makino H, Takata M, Miyata M, Nakamoto M, Burioka N and Shimizu E: Antibody-dependent cellular cytotoxicity mediated by cetuximab against lung cancer cell lines. Clin Cancer Res. 13:1552–1561. 2007. View Article : Google Scholar : PubMed/NCBI

12 

Chung KY, Shia J, Kemeny NE, Shah M, Schwartz GK, Tse A, Hamilton A, Pan D, Schrag D, Schwartz L, Klimstra DS, Fridman D, Kelsen DP and Saltz LB: Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol. 23:1803–1810. 2005. View Article : Google Scholar : PubMed/NCBI

13 

Jonker DJ, O’Callaghan CJ, Karapetis CS, Zalcberg JR, Tu D, Au HJ, Berry SR, Krahn M, Price T, Simes RJ, Tebbutt NC, van Hazel G, Wierzbicki R, Langer C and Moore MJ: Cetuximab for the treatment of colorectal cancer. N Engl J Med. 357:2040–2048. 2007. View Article : Google Scholar : PubMed/NCBI

14 

Sobrero AF, Maurel J, Fehrenbacher L, Scheithauer W, Abubakr YA, Lutz MP, Vega-Villegas ME, Eng C, Steinhauer EU, Prausova J, Lenz HJ, Borg C, Middleton G, Kröning H, Luppi G, Kisker O, Zubel A, Langer C, Kopit J and Burris HA III: EPIC: phase III trial of cetuximab plus irinotecan after fluoropyrimidine and oxaliplatin failure in patients with metastatic colorectal cancer. J Clin Oncol. 26:2311–2319. 2008. View Article : Google Scholar : PubMed/NCBI

15 

Bokemeyer C, Bondarenko I, Makhson A, Hartmann JT, Aparicio J, de Braud F, Donea S, Ludwig H, Schuch G, Stroh C, Loos AH, Zubel A and Koralewski P: Fluorouracil, leucovorin and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol. 27:663–671. 2009. View Article : Google Scholar : PubMed/NCBI

16 

Van Cutsem E, Köhne CH, Hitre E, Zaluski J, Chang Chien CR, Makhson A, D’Heans G, Pintér T, Lim R, Bodoky G, Roh JK, Folprecht G, Ruff P, Stroh C, Tejpar S, Schlichting M, Nippgen J and Rougier P: Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med. 360:1408–1417. 2009.PubMed/NCBI

17 

Vermorken JB, Mesia R, Rivera F, Remenar E, Kawecki A, Rottey S, Erfan J, Zabolotnyy D, Kienzer HR, Cupissol D, Peyrade F, Benasso M, Vynnychenko I, De Raucourt D, Bokemeyer C, Schueler A, Amellal N and Hitt R: Platinum-based chemotherapy plus cetuximab in head and neck cancer. N Engl J Med. 359:1116–1127. 2008. View Article : Google Scholar : PubMed/NCBI

18 

Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, Jones CU, Sur R, Raben D, Jassem J, Ove R, Kies MS, Baselga J, Youssoufian H, Amellal N, Rowinsky EK and Ang KK: Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 354:567–578. 2006. View Article : Google Scholar : PubMed/NCBI

19 

Herbst RS, Arquette M, Shin DM, Dicke K, Vokes EE, Azarnia N, Hong WK and Kies MS: Phase II multicenter study of the epidermal growth factor receptor antibody cetuximab and cisplatin for recurrent and refractory squamous cell carcinoma of the head and neck. J Clin Oncol. 23:5578–5587. 2005. View Article : Google Scholar : PubMed/NCBI

20 

Baselga J, Trigo JM, Bourhis J, Tortochaux J, Cortés-Funes H, Hitt R, Gascón P, Amellal N, Harstrick A and Eckardt A: Phase II multicenter study of the antiepidermal growth factor receptor monoclonal antibody cetuximab in combination with platinum-based chemotherapy in patients with platinum-refractory metastatic and/or recurrent squamous cell carcinoma of the head and neck. J Clin Oncol. 23:5568–5577. 2005.

21 

Burtness B, Goldwasser MA, Flood W, Mattar B and Forastiere AA; Eastern Cooperative Oncology Group. Phase III randomized trial of cisplatin plus placebo compared with cisplatin plus cetuximab in metastatic/recurrent head and neck cancer: an Eastern Cooperative Oncology Group study. J Clin Oncol. 23:8646–8654. 2005. View Article : Google Scholar : PubMed/NCBI

22 

Tejani MA, Cohen RB and Mehra T: The contribution of cetuximab in the treatment of recurrent and/or metastatic head and neck cancer. Biologics. 4:173–185. 2010.PubMed/NCBI

23 

Sok JC, Coppelli FM, Thomas SM, Lange MN, Xi S, Hunt JL, Freilino ML, Graner NW, Wikstrand CJ, Bigner DD, Gooding WE, Furnari FB and Grandis JR: Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targeting. Clin Cancer Res. 12:5064–5073. 2006. View Article : Google Scholar : PubMed/NCBI

24 

Chen LF, Cohen EE and Grandis JR: New strategies in head and neck cancer: understanding resistance to epidermal growth factor receptor inhibitors. Clin Cancer Res. 16:2489–2495. 2010. View Article : Google Scholar : PubMed/NCBI

25 

Kim SM, Kim JS, Kim JH, Yun CO, Kim EM, Kim HK, Solca F, Choi SY and Cho BC: Acquired resistance to cetuximab is mediated by increased PTEN instability and leads cross-resistance to gefitinib in HCC827 NSCLC cells. Cancer Lett. 296:150–159. 2010. View Article : Google Scholar : PubMed/NCBI

26 

Dunn EF, Iida M, Myers RA, Campbell DA, Hintz KA, Armstrong EA, Li C and Wheeler DL: Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab. Oncogene. 30:561–574. 2011. View Article : Google Scholar : PubMed/NCBI

27 

Ciardiello F, Bianco R, Caputo R, Caputo R, Damiano V, Troiani T, Melisi D, De Vita F, De Placido S, Bianco AR and Tortora G: Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy. Clin Cancer Res. 10:784–793. 2004. View Article : Google Scholar

28 

Wheeler DL, Iida M, Kruser TJ, Nechrebecki MM, Dunn EF, Armstrong EA, Huang S and Harari PM: Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab. Cancer Biol Ther. 8:696–703. 2009. View Article : Google Scholar : PubMed/NCBI

29 

Brand TM, Iida M and Wheeler DL: Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab. Cancer Biol Ther. 11:777–792. 2011. View Article : Google Scholar : PubMed/NCBI

30 

Yonesaka K1, Zejnullahu K, Okamoto I, Satoh T, Cappuzzo F, Souglakos J, Ercan D, Rogers A, Roncalli M, Takeda M, Fujisaka Y, Philips J, Shimizu T, Maenishi O, Cho Y, Sun J, Destro A, Taira K, Takeda K, Okabe T, Swanson J, Itoh H, Takada M, Lifshits E, Okuno K, Engelman JA, Shivdasani RA, Nishio K, Fukuoka M, Varella-Garcia M, Nakagawa K and Jänne PA: Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab. Sci Transl Med. 3:99ra862011.PubMed/NCBI

31 

Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ and Clarke MF: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 100:3983–3988. 2003. View Article : Google Scholar : PubMed/NCBI

32 

Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL and Wahl GM: Cancer stem cells - perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res. 66:9339–9344. 2006. View Article : Google Scholar

33 

Todaro M, Alea MP, Di Stefano AB, Cammareri P, Vermeulen L, Iovino F, Tripodo C, Russo A, Gulotta G, Medema JP and Stassi G: Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell. 1:389–402. 2007. View Article : Google Scholar : PubMed/NCBI

34 

Ma S, Lee TK, Zheng BJ, Chan KW and Guan XY: CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene. 27:1749–1758. 2008.

35 

Salmaggi A, Boiardi Ak, Gelati M, Russo A, Calatozzolo C, Ciusani E, Sciacca FL, Ottolina A, Parati EA, La Porta C, Alessandri G, Marras C, Croci D and De Rossi M: Glioblastoma-derived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype. Glia. 54:850–860. 2006. View Article : Google Scholar

36 

Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ and Wicha MS: In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 17:1253–1270. 2003. View Article : Google Scholar : PubMed/NCBI

37 

Thomas SM and Grandis JR: Pharmacokinetic and pharmacodynamics properties of EGFR inhibitors under clinical investigation. Cancer Treat Rev. 30:255–268. 2004. View Article : Google Scholar : PubMed/NCBI

38 

Pirker R, Pereira JR, von Pawel J, Krzakowski M, Ramlau R, Park K, de Marinis F, Eberhardt WE, Paz-Ares L, Störkel S, Shumacher KM, von Heydebreck A, Celik I and O’Byrne KJ: EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer: analysis of data from the phase 3 FLEX study. Lancet Oncol. 13:33–42. 2012. View Article : Google Scholar : PubMed/NCBI

39 

Wheeler DL, Huang S, Kruser TJ, Nechrebecki MM, Armstrong EA, Benavente S, Gondi V, Hsu KT and Harari PM: Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members. Oncogene. 27:3944–3956. 2008. View Article : Google Scholar : PubMed/NCBI

40 

Nevo J, Mattila E, Pellinen T, Yamamoto DL, Sara H, Iljin K, Kallioniemi O, Bono P, Heikkilä P, Joensuu H, Wärri A and Ivaska J: Mammary-derived growth inhibitor alters traffic of EGFR and induces a novel form of cetuximab resistance. Clin Cancer Res. 15:6570–6581. 2009. View Article : Google Scholar : PubMed/NCBI

41 

Hatakeyama H, Cheng H, Wirth P, Counsell A, Marcrom SR, Wood CB, Pohlmann PR, Gilbert J, Murphy B, Yarbrough WG, Wheeler DL, Harari PM, Guo Y, Shyr Y, Slebos RJ and Chung CH: Regulation of heparin-binding EGF-like growth factor by miR-212 and acquired cetuximab-resistance in head and neck squamous cell carcinoma. PLoS One. 5:e127022010. View Article : Google Scholar : PubMed/NCBI

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August 2014
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Copy and paste a formatted citation
APA
Ohnishi, Y., Minamino, Y., Kakudo, K., & Nozaki, M. (2014). Resistance of oral squamous cell carcinoma cells to cetuximab is associated with EGFR insensitivity and enhanced stem cell-like potency. Oncology Reports, 32, 780-786. https://doi.org/10.3892/or.2014.3258
MLA
Ohnishi, Y., Minamino, Y., Kakudo, K., Nozaki, M."Resistance of oral squamous cell carcinoma cells to cetuximab is associated with EGFR insensitivity and enhanced stem cell-like potency". Oncology Reports 32.2 (2014): 780-786.
Chicago
Ohnishi, Y., Minamino, Y., Kakudo, K., Nozaki, M."Resistance of oral squamous cell carcinoma cells to cetuximab is associated with EGFR insensitivity and enhanced stem cell-like potency". Oncology Reports 32, no. 2 (2014): 780-786. https://doi.org/10.3892/or.2014.3258