Abstract
Purpose
The clinical outcome of head and neck squamous cell carcinoma (HNSCC) remains poor, partly due to the presence of resistant cancer stem cells (CSCs) which are responsible of recurrences. CSCs have low EGFR expression and, conversely, overexpress the anti-apoptotic Bcl-2 protein, which is involved in resistance to apoptosis and the invasion/migration capacities of tumour cells.
Methods
The combination therapy of ABT-199, a Bcl-2 inhibitor, cetuximab an EGFR inhibitor, and radiation using an HNSCC model (SQ20B cell line) and its corresponding CSC subpopulation were evaluated in vitro (2D/3D cell proliferation; invasion/migration and apoptosis using videomicroscopy) and in vivo.
Results
Cetuximab strongly inhibited 2D and 3D cell proliferation, as well as invasion/migration, only in non-CSC-SQ20B cells, whereas ABT-199 selectively inhibited these mechanisms in SQ20B/CSCs. The combination of irradiation + cetuximab + ABT-199 increased the inhibition of the 2D and 3D cell proliferation, invasion/migration, and resistance to apoptosis in both cell sub-populations. In addition, in a nude mouse model with heterotopic tumour xenograft, a treatment combining cetuximab + ABT-199 with fractional irradiation strongly delayed the tumour growth and increased in vivo lifespan without side effects.
Conclusion
Based on the present results, this triple combination therapy may represent a new opportunity for testing in clinical trials, particularly in locally advanced HNSCC.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
References
Abdullah LN, Chow EK-H (2013) Mechanisms of chemoresistance in cancer stem cells. Clin Transl Med 2:3. https://doi.org/10.1186/2001-1326-2-3
Adhya AK, Srinivasan R, Patel FD (2006) Radiation therapy induced changes in apoptosis and its major regulatory proteins, Bcl-2, Bcl-XL, and Bax, in locally advanced invasive squamous cell carcinoma of the cervix. Int J Gynecol Pathol Off J Int Soc Gynecol Pathol 25:281–287. https://doi.org/10.1097/01.pgp.0000215292.99996.44
Baumann M, Krause M, Hill R (2008) Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer 8:545–554. https://doi.org/10.1038/nrc2419
Bertrand G, Maalouf M, Boivin A et al (2014) Targeting head and neck cancer stem cells to overcome resistance to photon and carbon ion radiation. Stem Cell Rev Rep 10:114–126. https://doi.org/10.1007/s12015-013-9467-y
Bonner JA, Raisch KP, Trummell HQ et al (2000) Enhanced apoptosis with combination C225/radiation treatment serves as the impetus for clinical investigation in head and neck cancers. J Clin Oncol Off J Am Soc Clin Oncol 18:47S-53S
Bonner JA, Harari PM, Giralt J et al (2006) Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 354:567–578. https://doi.org/10.1056/NEJMoa053422
Bonner JA, Harari PM, Giralt J et al (2010) Radiotherapy plus cetuximab for locoregionally advanced head and neck cancer: 5-year survival data from a phase 3 randomised trial, and relation between cetuximab-induced rash and survival. Lancet Oncol 11:21–28. https://doi.org/10.1016/S1470-2045(09)70311-0
Bourhis J (2019) Double-blind Randomized Phase 2 Results Comparing Concurrent High-dose Cisplatin Chemorradiation (CRT) plus Debio 1143 or Placebo In High-risk Patients with Locally Advanced Squamous Cell Carcinoma of the Head and Neck (SCCHN): A GORTEC Study
Brand TM, Iida M, Wheeler DL (2011) Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab. Cancer Biol Ther 11:777–792. https://doi.org/10.4161/cbt.11.9.15050
Braunholz D, Saki M, Niehr F et al (2016) Spheroid culture of head and neck cancer cells reveals an important role of EGFR signalling in anchorage independent survival. PLoS ONE 11:e0163149. https://doi.org/10.1371/journal.pone.0163149
Caldas-Lopes E, Gomez-Arteaga A, Guzman ML (2019) Approaches to targeting cancer stem cells in solid tumors. Curr Stem Cell Res Ther 14:421–427. https://doi.org/10.2174/1574888X14666190222164429
Castedo M, Perfettini J-L, Roumier T et al (2004) Cell death by mitotic catastrophe: a molecular definition. Oncogene 23:2825–2837. https://doi.org/10.1038/sj.onc.1207528
Chen L, Willis SN, Wei A et al (2005) Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 17:393–403. https://doi.org/10.1016/j.molcel.2004.12.030
Chung CH, Ely K, McGavran L et al (2006) Increased epidermal growth factor receptor gene copy number is associated with poor prognosis in head and neck squamous cell carcinomas. J Clin Oncol Off J Am Soc Clin Oncol 24:4170–4176. https://doi.org/10.1200/JCO.2006.07.2587
Cohen EEW (2006) Role of epidermal growth factor receptor pathway-targeted therapy in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck. J Clin Oncol Off J Am Soc Clin Oncol 24:2659–2665. https://doi.org/10.1200/JCO.2005.05.4577
Cramer JD, Burtness B, Le QT, Ferris RL (2019) The changing therapeutic landscape of head and neck cancer. Nat Rev Clin Oncol 16:669–683. https://doi.org/10.1038/s41571-019-0227-z
Del Gaizo MV, Letai A (2013) BH3 profiling–measuring integrated function of the mitochondrial apoptotic pathway to predict cell fate decisions. Cancer Lett 332:202–205. https://doi.org/10.1016/j.canlet.2011.12.021
Deng X, Gao F, May WS (2003) Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS. Blood 102:3179–3185. https://doi.org/10.1182/blood-2003-04-1027
dos Santos LV, Carvalho AL (2011) Bcl-2 targeted-therapy for the treatment of head and neck squamous cell carcinoma. Recent Patents Anticancer Drug Discov 6:45–57. https://doi.org/10.2174/157489211793980042
Edlich F (2018) BCL-2 proteins and apoptosis: recent insights and unknowns. Biochem Biophys Res Commun 500:26–34. https://doi.org/10.1016/j.bbrc.2017.06.190
Fischer K, Al-Sawaf O, Bahlo J et al (2019) Venetoclax and obinutuzumab in patients with CLL and coexisting conditions. N Engl J Med 380:2225–2236. https://doi.org/10.1056/NEJMoa1815281
Gallo O, Boddi V, Calzolari A et al (1996) bcl-2 protein expression correlates with recurrence and survival in early stage head and neck cancer treated by radiotherapy. Clin Cancer Res Off J Am Assoc Cancer Res 2:261–267
Gilormini M, Wozny A-S, Battiston-Montagne P et al (2016b) Isolation and characterization of a head and neck squamous cell carcinoma subpopulation having stem cell characteristics. J Vis Exp JoVE. https://doi.org/10.3791/53958
Gilormini M, Malesys C, Armandy E et al (2016a) Preferential targeting of cancer stem cells in the radiosensitizing effect of ABT-737 on HNSCC. Oncotarget 7:16731–16744. https://doi.org/10.18632/oncotarget.7744
Gong Y, Somwar R, Politi K et al (2007) Induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors in mutant EGFR-dependent lung adenocarcinomas. PLoS Med 4:e294. https://doi.org/10.1371/journal.pmed.0040294
Grønhøj C, Jakobsen KK, Jensen DH et al (2018) Pattern of and survival following loco-regional and distant recurrence in patients with HPV+ and HPV- oropharyngeal squamous cell carcinoma: a population-based study. Oral Oncol 83:127–133. https://doi.org/10.1016/j.oraloncology.2018.06.012
Guy J-B, Méry B, Ollier E et al (2017) Dual “mAb” HER family blockade in head and neck cancer human cell lines combined with photon therapy. Sci Rep 7:12207. https://doi.org/10.1038/s41598-017-12367-7
Han Z, Liang J, Li Y, He J (2019) Drugs and clinical approaches targeting the antiapoptotic protein: a review. BioMed Res Int 2019:1212369. https://doi.org/10.1155/2019/1212369
Jones AK, Freise KJ, Agarwal SK et al (2016) Clinical predictors of venetoclax pharmacokinetics in chronic lymphocytic leukemia and non-Hodgkin’s lymphoma patients: a pooled population pharmacokinetic analysis. AAPS J 18:1192–1202. https://doi.org/10.1208/s12248-016-9927-9
Kalyankrishna S, Grandis JR (2006) Epidermal growth factor receptor biology in head and neck cancer. J Clin Oncol Off J Am Soc Clin Oncol 24:2666–2672. https://doi.org/10.1200/JCO.2005.04.8306
Kater AP, Levin M-D, Niemann CU (2019) Ibrutinib and venetoclax for first-line treatment of CLL. N Engl J Med 381:788–789. https://doi.org/10.1056/NEJMc1908754
Ku B, Liang C, Jung JU, Oh B-H (2011) Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX. Cell Res 21:627–641. https://doi.org/10.1038/cr.2010.149
Kumar P, Ning Y, Polverini PJ (2008) Endothelial cells expressing Bcl-2 promotes tumor metastasis by enhancing tumor angiogenesis, blood vessel leakiness and tumor invasion. Lab Investig J Tech Methods Pathol 88:740–749. https://doi.org/10.1038/labinvest.2008.46
La Fleur L, Johansson A-C, Roberg K (2012) A CD44high/EGFRlow subpopulation within head and neck cancer cell lines shows an epithelial-mesenchymal transition phenotype and resistance to treatment. PLoS ONE 7:e44071. https://doi.org/10.1371/journal.pone.0044071
Lamouille S, Xu J, Derynck R (2014) Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 15:178–196. https://doi.org/10.1038/nrm3758
Lee CM, Tannock IF (2010) The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors. BMC Cancer 10:255. https://doi.org/10.1186/1471-2407-10-255
Maalouf M, Alphonse G, Colliaux A et al (2009) Different mechanisms of cell death in radiosensitive and radioresistant p53 mutated head and neck squamous cell carcinoma cell lines exposed to carbon ions and x-rays. Int J Radiat Oncol Biol Phys 74:200–209. https://doi.org/10.1016/j.ijrobp.2009.01.012
Matzinger O, Viertl D, Tsoutsou P et al (2015) The radiosensitizing activity of the SMAC-mimetic, Debio 1143, is TNFα-mediated in head and neck squamous cell carcinoma. Radiother Oncol J Eur Soc Ther Radiol Oncol 116:495–503. https://doi.org/10.1016/j.radonc.2015.05.017
Moncharmont C, Guy J-B, Wozny A-S et al (2016) Carbon ion irradiation withstands cancer stem cells’ migration/invasion process in Head and Neck Squamous Cell Carcinoma (HNSCC). Oncotarget 7:47738–47749. https://doi.org/10.18632/oncotarget.10281
Noordhuis MG, Eijsink JJH, Ten Hoor KA et al (2009) Expression of epidermal growth factor receptor (EGFR) and activated EGFR predict poor response to (chemo)radiation and survival in cervical cancer. Clin Cancer Res Off J Am Assoc Cancer Res 15:7389–7397. https://doi.org/10.1158/1078-0432.CCR-09-1149
Pan R, Hogdal LJ, Benito JM et al (2014) Selective BCL-2 inhibition by ABT-199 causes on-target cell death in acute myeloid leukemia. Cancer Discov 4:362–375. https://doi.org/10.1158/2159-8290.CD-13-0609
Roberts AW, Davids MS, Pagel JM et al (2016) Targeting BCL2 with venetoclax in relapsed chronic lymphocytic leukemia. N Engl J Med 374:311–322. https://doi.org/10.1056/NEJMoa1513257
Seymour JF, Kipps TJ, Eichhorst B et al (2018) Venetoclax-rituximab in relapsed or refractory chronic lymphocytic leukemia. N Engl J Med 378:1107–1120. https://doi.org/10.1056/NEJMoa1713976
Swick AD, Prabakaran PJ, Miller MC et al (2017) Cotargeting mTORC and EGFR signaling as a therapeutic strategy in HNSCC. Mol Cancer Ther 16:1257–1268. https://doi.org/10.1158/1535-7163.MCT-17-0115
Tsujimoto Y, Yunis J, Onorato-Showe L et al (1984) Molecular cloning of the chromosomal breakpoint of B-cell lymphomas and leukemias with the t(11;14) chromosome translocation. Science 224:1403–1406. https://doi.org/10.1126/science.6610211
Um H-D (2016) Bcl-2 family proteins as regulators of cancer cell invasion and metastasis: a review focusing on mitochondrial respiration and reactive oxygen species. Oncotarget 7:5193–5203. https://doi.org/10.18632/oncotarget.6405
Wang J, Valdez A, Chen Y (2017) Evaluation of automated Wes system as an analytical and characterization tool to support monoclonal antibody drug product development. J Pharm Biomed Anal 139:263–268. https://doi.org/10.1016/j.jpba.2016.12.024
Wang Y, Wang Y, Fan X et al (2018) ABT-199-mediated inhibition of Bcl-2 as a potential therapeutic strategy for nasopharyngeal carcinoma. Biochem Biophys Res Commun 503:1214–1220. https://doi.org/10.1016/j.bbrc.2018.07.027
Wozny A-S, Lauret A, Battiston-Montagne P et al (2017) Differential pattern of HIF-1α expression in HNSCC cancer stem cells after carbon ion or photon irradiation: one molecular explanation of the oxygen effect. Br J Cancer 116:1340–1349. https://doi.org/10.1038/bjc.2017.100
Wozny A-S, Vares G, Alphonse G et al (2019) ROS production and distribution: a new paradigm to explain the differential effects of x-ray and carbon ion irradiation on cancer stem cell migration and invasion. Cancers. https://doi.org/10.3390/cancers11040468
Acknowledgements
We thank all participants in the experiments and acknowledge the contribution of the flow cytometry platform of the SFR BioSciencesGerland-Lyon Sud (UMS3444/US8).
Funding
This work was supported by the LABEX PRIMES (ANR-11-LABX-0063) of Université de Lyon, within the program “Investissements d’Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). It was also financed by La Ligue Contre le Cancer Comité de la Loire.
Author information
Authors and Affiliations
Contributions
JBG, SE, NM, and CRL designed the study. JBG, SE, NV, and CM did the 2D and 3D experiments. JBG, SE, SL, GA, and CM performed the in vivo experiments. JBG, SE, MAG, AG, ASW, CM, and GA did the collection and assembly of the data. Resources and funding were obtained by CRL, NN, and JBG. The original writing draft was prepared by JB and SE, and corrected by DA, GA, NM, and CRL.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
432_2021_3593_MOESM1_ESM.pdf
Supplementary file1 (PDF 153 KB) Supplementary Data 1. Statistical analysis of cell proliferation measured at 120h in SQ20B cells (A) and SQ20B/CSCs (B) exposed to the various treatments. *P < 0.05; **P < 0.01; ***P < 0.001.
432_2021_3593_MOESM2_ESM.pdf
Supplementary file2 (PDF 178 KB) Supplementary Data 2. Microscopic observation (10×) of the spheroid growth of SQ20B cells and SQ20B/CSCs. (A) SQ20B 3D-spheroid growth observed as red fluorescence by phase contrast video microscopy at 2, 5, 7, 10 and 14 days. (B) SQ20B 3D-spheroid growth observed as red fluorescence by video microscopy at 2, 5, 7, 10 and 14 days.(C)SQ20B/CSC 3D-spheroid growth observed as red fluorescence and phase contrast video microscopy at 2, 5, 7, 10 and 14 days. (D)SQ20B/CSC 3D-spheroid growth observed in 10× red fluorescence video microscopy at 2, 5, 7, 10 and 14 days.
432_2021_3593_MOESM3_ESM.pdf
Supplementary file3 (PDF 154 KB) Supplementary Data 3. Statistical analysis of 3D-spheroid growth in SQ20B cells (A) and SQ20B/CSCs (B) exposed to the various treatments.Cell 3D-spheroid growth was measured as the mean fluorescence intensity (RCU) at 10 days. *P < 0.05; **P < 0.01; ***P < 0.001.
432_2021_3593_MOESM4_ESM.pdf
Supplementary file4 (PDF 107 KB) Supplementary Data 4. Statistical analysis of caspase 3/7 activation in SQ20B cells (A) and SQ20B/CSCs (B) exposed to the various treatments. Cellular apoptosis was measured at 120h and is expressed as a percentage of activated caspase 3/7. *P < 0.05; **P < 0.01; ***P < 0.001.
432_2021_3593_MOESM5_ESM.pdf
Supplementary file5 (PDF 151 KB) Supplementary Data 5. Statistical analysis of migration assays of SQ20B cells (A) and SQ20B/CSCs (B) exposed to the various treatments. Cell migration was measured by relative wound density at 24 h. *P < 0.05; **P < 0.01; ***P < 0.001.
432_2021_3593_MOESM6_ESM.pdf
Supplementary file6 (PDF 151 KB) Supplementary Data 6. Statistical analysis of invasion assays of SQ20B cells (A) and SQ20B/CSC (B) exposed to the various treatments. Cell invasion was measured by relative wound density at 48 h. *P < 0.05; **P < 0.01; ***P < 0.001.
Rights and permissions
About this article
Cite this article
Guy, JB., Espenel, S., Louati, S. et al. Combining radiation to EGFR and Bcl-2 blockade: a new approach to target cancer stem cells in head and neck squamous cell carcinoma. J Cancer Res Clin Oncol 147, 1905–1916 (2021). https://doi.org/10.1007/s00432-021-03593-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00432-021-03593-8