JNCI J Natl Cancer Inst (2015) 107(2): dju440 doi: 10.1093/jnci/dju440 First published online February 6, 2015 Editorial
editorial Towards an Integrated Understanding of Epidermal Growth Factor Receptor Biology for Radiation Therapy: Integrins Enter Henning Willers, Theodore S. Hong Affiliation of authors: Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts (HW, TSH).
It is clear that a huge body of preclinical data on combined radiation and radiosensitizing targeted drugs has not translated into an adequate number of successful radiation oncology trials. In light of this discordance, recent National Cancer Institute (NCI)–Radiation Therapy Oncology (RTOG) guidelines have emphasized the need to develop better preclinical models (1). The epidermal growth factor receptor (EGFR) has been intensely studied as a target for radiation therapy for more than a decade, but despite their considerable promise the benefit of EGFR-directed radiosensitizers in clinical practice remains to be fully realized (2–4). It is tempting to speculate that we still do not know EGFR biology well enough, which bears some similarity to KRAS (5), and this prevents us from optimally exploiting EGFR for radiation therapy. Better preclinical models that provide an integrated understanding of EGFR biology and account for clinical tumor heterogeneity are urgently needed. In this issue (6), Nils Cordes and colleagues tackled the important clinical question of whether EGFR targeting alone is insufficient to achieve complete radiosensitization and local tumor control in head and neck squamous cell carcinoma (HNSCC). Supported by emerging data implicating integrins as promising anticancer drug targets and links between integrins and EGFR (7), the authors hypothesized that EGFR and integrin β1 promote mutual survival bypass signaling in HNSCC treated with concurrent radiation and single targeted drug, ie, integrin β1–directed AIIB2 antibody or EGFR monoclonal antibody cetuximab. Through a phospho-proteomics approach the authors identified activation of mitogen-activated protein kinase kinase (MEK)/extracellularsignal-regulated kinase (ERK) signaling in HNSCC in which integrin β1 function was disrupted, suggesting a potential link to EGFR. In a panel of 10 HNSCC cell lines grown in extracellular matrix-enriched 3D cultures, cetuximab radiosensitized five cell lines while AIIB2 radiosensitized another three cell lines. Combined antibody treatment led to more substantial radiosensitization than inhibition of either receptor alone in these eight cell lines. The in vitro data were validated in xenograft models. Strikingly, for a representative susceptible cell line, the local tumor control probability using a single
radiation treatment was 100% for combined cetuximab/AIIB2 but only 30% to 60% for individual antibody therapies. To better understand the effects of combined receptor blockade on cellular signaling, the authors then carried out a large-scale phospho-proteomic analysis to characterize signaling network perturbations as a result of receptor inhibitions. An elegant bioinformatics approach confirmed the superiority of combined receptor blockade over single blockade. Phospho-proteomic analysis also implicated focal adhesion kinase (FAK) as a critical signaling node that promoted the repair of radiation-induced DNA double-strand breaks (DSB) and cell survival downstream of EGFR-MEK-ERK and integrin β1. Consistent with this finding, exogenous overexpression of a constitutively active form of FAK completely abrogated the radiosensitizing effects of combined cetuximab/AIIB2. Cumulatively, the data suggest a novel model in which integrin β1-FAK signaling can support DSB repair and survival in the absence of functional EGFR (Figure 1). This raises several interesting questions: Do FAK inhibitors, which are in clinical trials (8), have the same radiosensitizing effect as combined EGFR/integrin β1 blockade? Is the unexpected position of FAK downstream of ERK indicative of a proximal, and perhaps nuclear, role in the cellular DNA damage response? Given the reported links between integrins and stem cells (9,10), might integrin β1 promote stemness and cancer stem cell (CSC) survival during combined fractionated radiation and cetuximab treatment? And to clinically translate combined EGFR-integrin β1 targeting and radiation, will predictive biomarkers be required? Because the authors observed radiosensitization by combined receptor targeting in nine of 10 cell lines, the answer to this is perhaps “no.” On the other hand, the clinically relevant genomic heterogeneity of HNSCC is likely larger than can be captured with 10 cell lines (11). This relates to a major question in radiation oncology: How many cancer cell lines are needed for preclinical assessment of radiosensitizing agents, such as an EGFR-targeted agent? Is it 10 or 40 (12), or is it more than 100 that are evidently necessary to match drug-alone sensitivities to rare genomic alterations (13)?
Accepted: December 09, 2014 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected].
1 of 2
Downloaded from http://jnci.oxfordjournals.org/ by guest on June 4, 2015
Correspondence to: Henning Willers, MD, Laboratory of Cellular and Molecular Radiation Oncology, Massachusetts General Hospital Cancer Center, 149 13th Street, Charlestown, MA 02129 (e-mail: [email protected]).
Willers et al. | 2 of 2
Radiation induces numerous stress and DNA damage responses. Multitarget or intentionally “dirty” inhibitors, therefore, may be best suited to achieve full radiosensitization and perhaps counter adaptive CSC responses during fractionated radiation therapy. After all, this remains the overarching goal of curative radiation therapy—to eliminate the last surviving CSC in order to locally control the tumor (14).
References
Traditionally, radiation/drug combinations have been studied in a very limited number of human or even nonhuman cell lines, conforming to a “one size fits all” philosophy. However, any radiosensitizing effects in one or few cell lines may not be representative of efficacy in an unselected larger number of genomically heterogeneous tumors, which may only be revealed when the agent under study has entered clinical trials. In our opinion, the answer to the question of how many genomically diverse cancer cell lines will be routinely required to robustly assess the fraction that can be radiosensitized and to correlate potential predictive biomarkers with radiosensitization is likely going to be higher than we might like (perhaps as high as 50–100). With regard to biomarkers, the authors suggested that dephosphorylated FAK may be useful to identify HNSCC in which EGFR/integrin β1 inhibition is effective. However, absence of a phospho-signal could be difficult to implement as a robust clinical test. Another promising avenue is the assessment of DSB-associated protein foci in tumor nuclei as a functional biomarker of radiosensitizing effect (γ-H2AX or 53BP1). This would require rebiopsy following combined radiation/drug treatment or ex vivo treatment of tumor biopsies from patients before starting protocol therapy. Nils Cordes and colleagues (6) are applauded for vastly improving preclinical modeling of HNSCC treatment responses by using a larger number of cell lines than traditionally employed, by growing cells in a more physiological matrixenriched 3D culture, and by introducing a bioinformatics approach to tackle the complex biology of EGFR and associated signaling networks. Findings of their study are perhaps most notable for highlighting the need for a multitarget approach in radiation oncology. For the longest time, we have strived to identify individual molecular targets of radiosensitization for a given drug/cancer combination. Most likely, this is overly optimistic.
Downloaded from http://jnci.oxfordjournals.org/ by guest on June 4, 2015
Figure 1. Model of epidermal growth factor receptor’s (EGFR’s) role in radioresistance. New data support a role of focal adhesion kinase (FAK) in promoting DNA double-strand break repair and cell survival following treatment with ionizing radiation. In this model, an ERK-FAK complex acts downstream of MEK and integrin β1, at least in head and neck squamous cell carcinoma. Previous data have suggested roles of DNA-PK, PKC, and MEK in EGFR-mediated radioresistance. DSB = double-strand break; EGFR = epidermal growth factor receptor; FAK = focal adhesion kinase.
1. Lawrence YR, Vikram B, Dignam JJ, et al. NCI-RTOG translational program strategic guidelines for the earlystage development of radiosensitizers. J Natl Cancer Inst. 2013;105(1):11–24. 2. Martins RG, Parvathaneni U, Bauman JE, et al. Cisplatin and radiotherapy with or without erlotinib in locally advanced squamous cell carcinoma of the head and neck: a randomized phase II trial. J Clin Oncol. 2013;31(11):1415–1421. 3. Ang KK, Zhang Q, Rosenthal DI, et al. Randomized Phase III Trial of Concurrent Accelerated Radiation Plus Cisplatin With or Without Cetuximab for Stage III to IV Head and Neck Carcinoma: RTOG 0522. J Clin Oncol. 2014; In press. 4. Bradley J, Masters GA, Hu C, et al. An intergroup randomized phase III comparison of standard-dose (60 Gy) versus highdose (74 Gy) chemoradiotherapy (CRT) +/- cetuximab (cetux) for stage III non-small cell lung cancer (NSCLC): Results on cetux from RTOG 0617. In: 15th World Conference on Lung Cancer, 2013. Sydney, Australia; 2013. 5. Stephen AG, Esposito D, Bagni RK, McCormick F. Dragging ras back in the ring. Cancer Cell. 2014;25(3):272–281. 6. Eke I, Zscheppang K, Dickreuter E, et al. Simultaneous β1 integrin-EGFR targeting and radiosensitization of human head and neck cancer. J Natl Cancer Inst. 2015;107(2):dju419 doi:10.1093/jnci/dju419. 7. Goodman SL, Picard M. Integrins as therapeutic targets. Trends Pharmacol Sci. 2012;33(7):405–412. 8. Lee BY, Timpson P, Horvath LG, Daly RJ. FAK Signaling in Human Cancer as a Target for Therapeutics. Pharmacol Ther. 2014; In press. 9. Seguin L, Kato S, Franovic A, et al. An integrin beta(3)-KRASRalB complex drives tumour stemness and resistance to EGFR inhibition. Nat Cell Biol. 2014;16(5):457–468. 10. Brizzi MF, Tarone G, Defilippi P. Extracellular matrix, integrins, and growth factors as tailors of the stem cell niche. Curr Opin Cell Biol. 2012;24(5):645–651. 11. Stransky N, Egloff AM, Tward AD, et al. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333(6046):1157–1160. 12. Wang M, Kern AM, Hulskotter M, et al. EGFR-mediated chromatin condensation protects KRAS-mutant cancer cells against ionizing radiation. Cancer Res. 2014;74(10):2825–2834. 13. McDermott U, Sharma SV, Dowell L, et al. Identification of genotype-correlated sensitivity to selective kinase inhibitors by using high-throughput tumor cell line profiling. Proc Natl Acad Sci U S A. 2007;104(50):19936–19941. 14. Krause M, Yaromina A, Eicheler W, Koch U, Baumann M. Cancer stem cells: targets and potential biomarkers for radiotherapy. Clin Cancer Res. 2011;17(23):7224–7229.
editorial Towards an Integrated Understanding of Epidermal Growth Factor Receptor Biology for Radiation Therapy: Integrins Enter Henning Willers, Theodore S. Hong Affiliation of authors: Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts (HW, TSH).
It is clear that a huge body of preclinical data on combined radiation and radiosensitizing targeted drugs has not translated into an adequate number of successful radiation oncology trials. In light of this discordance, recent National Cancer Institute (NCI)–Radiation Therapy Oncology (RTOG) guidelines have emphasized the need to develop better preclinical models (1). The epidermal growth factor receptor (EGFR) has been intensely studied as a target for radiation therapy for more than a decade, but despite their considerable promise the benefit of EGFR-directed radiosensitizers in clinical practice remains to be fully realized (2–4). It is tempting to speculate that we still do not know EGFR biology well enough, which bears some similarity to KRAS (5), and this prevents us from optimally exploiting EGFR for radiation therapy. Better preclinical models that provide an integrated understanding of EGFR biology and account for clinical tumor heterogeneity are urgently needed. In this issue (6), Nils Cordes and colleagues tackled the important clinical question of whether EGFR targeting alone is insufficient to achieve complete radiosensitization and local tumor control in head and neck squamous cell carcinoma (HNSCC). Supported by emerging data implicating integrins as promising anticancer drug targets and links between integrins and EGFR (7), the authors hypothesized that EGFR and integrin β1 promote mutual survival bypass signaling in HNSCC treated with concurrent radiation and single targeted drug, ie, integrin β1–directed AIIB2 antibody or EGFR monoclonal antibody cetuximab. Through a phospho-proteomics approach the authors identified activation of mitogen-activated protein kinase kinase (MEK)/extracellularsignal-regulated kinase (ERK) signaling in HNSCC in which integrin β1 function was disrupted, suggesting a potential link to EGFR. In a panel of 10 HNSCC cell lines grown in extracellular matrix-enriched 3D cultures, cetuximab radiosensitized five cell lines while AIIB2 radiosensitized another three cell lines. Combined antibody treatment led to more substantial radiosensitization than inhibition of either receptor alone in these eight cell lines. The in vitro data were validated in xenograft models. Strikingly, for a representative susceptible cell line, the local tumor control probability using a single
radiation treatment was 100% for combined cetuximab/AIIB2 but only 30% to 60% for individual antibody therapies. To better understand the effects of combined receptor blockade on cellular signaling, the authors then carried out a large-scale phospho-proteomic analysis to characterize signaling network perturbations as a result of receptor inhibitions. An elegant bioinformatics approach confirmed the superiority of combined receptor blockade over single blockade. Phospho-proteomic analysis also implicated focal adhesion kinase (FAK) as a critical signaling node that promoted the repair of radiation-induced DNA double-strand breaks (DSB) and cell survival downstream of EGFR-MEK-ERK and integrin β1. Consistent with this finding, exogenous overexpression of a constitutively active form of FAK completely abrogated the radiosensitizing effects of combined cetuximab/AIIB2. Cumulatively, the data suggest a novel model in which integrin β1-FAK signaling can support DSB repair and survival in the absence of functional EGFR (Figure 1). This raises several interesting questions: Do FAK inhibitors, which are in clinical trials (8), have the same radiosensitizing effect as combined EGFR/integrin β1 blockade? Is the unexpected position of FAK downstream of ERK indicative of a proximal, and perhaps nuclear, role in the cellular DNA damage response? Given the reported links between integrins and stem cells (9,10), might integrin β1 promote stemness and cancer stem cell (CSC) survival during combined fractionated radiation and cetuximab treatment? And to clinically translate combined EGFR-integrin β1 targeting and radiation, will predictive biomarkers be required? Because the authors observed radiosensitization by combined receptor targeting in nine of 10 cell lines, the answer to this is perhaps “no.” On the other hand, the clinically relevant genomic heterogeneity of HNSCC is likely larger than can be captured with 10 cell lines (11). This relates to a major question in radiation oncology: How many cancer cell lines are needed for preclinical assessment of radiosensitizing agents, such as an EGFR-targeted agent? Is it 10 or 40 (12), or is it more than 100 that are evidently necessary to match drug-alone sensitivities to rare genomic alterations (13)?
Accepted: December 09, 2014 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected].
1 of 2
Downloaded from http://jnci.oxfordjournals.org/ by guest on June 4, 2015
Correspondence to: Henning Willers, MD, Laboratory of Cellular and Molecular Radiation Oncology, Massachusetts General Hospital Cancer Center, 149 13th Street, Charlestown, MA 02129 (e-mail: [email protected]).
Willers et al. | 2 of 2
Radiation induces numerous stress and DNA damage responses. Multitarget or intentionally “dirty” inhibitors, therefore, may be best suited to achieve full radiosensitization and perhaps counter adaptive CSC responses during fractionated radiation therapy. After all, this remains the overarching goal of curative radiation therapy—to eliminate the last surviving CSC in order to locally control the tumor (14).
References
Traditionally, radiation/drug combinations have been studied in a very limited number of human or even nonhuman cell lines, conforming to a “one size fits all” philosophy. However, any radiosensitizing effects in one or few cell lines may not be representative of efficacy in an unselected larger number of genomically heterogeneous tumors, which may only be revealed when the agent under study has entered clinical trials. In our opinion, the answer to the question of how many genomically diverse cancer cell lines will be routinely required to robustly assess the fraction that can be radiosensitized and to correlate potential predictive biomarkers with radiosensitization is likely going to be higher than we might like (perhaps as high as 50–100). With regard to biomarkers, the authors suggested that dephosphorylated FAK may be useful to identify HNSCC in which EGFR/integrin β1 inhibition is effective. However, absence of a phospho-signal could be difficult to implement as a robust clinical test. Another promising avenue is the assessment of DSB-associated protein foci in tumor nuclei as a functional biomarker of radiosensitizing effect (γ-H2AX or 53BP1). This would require rebiopsy following combined radiation/drug treatment or ex vivo treatment of tumor biopsies from patients before starting protocol therapy. Nils Cordes and colleagues (6) are applauded for vastly improving preclinical modeling of HNSCC treatment responses by using a larger number of cell lines than traditionally employed, by growing cells in a more physiological matrixenriched 3D culture, and by introducing a bioinformatics approach to tackle the complex biology of EGFR and associated signaling networks. Findings of their study are perhaps most notable for highlighting the need for a multitarget approach in radiation oncology. For the longest time, we have strived to identify individual molecular targets of radiosensitization for a given drug/cancer combination. Most likely, this is overly optimistic.
Downloaded from http://jnci.oxfordjournals.org/ by guest on June 4, 2015
Figure 1. Model of epidermal growth factor receptor’s (EGFR’s) role in radioresistance. New data support a role of focal adhesion kinase (FAK) in promoting DNA double-strand break repair and cell survival following treatment with ionizing radiation. In this model, an ERK-FAK complex acts downstream of MEK and integrin β1, at least in head and neck squamous cell carcinoma. Previous data have suggested roles of DNA-PK, PKC, and MEK in EGFR-mediated radioresistance. DSB = double-strand break; EGFR = epidermal growth factor receptor; FAK = focal adhesion kinase.
1. Lawrence YR, Vikram B, Dignam JJ, et al. NCI-RTOG translational program strategic guidelines for the earlystage development of radiosensitizers. J Natl Cancer Inst. 2013;105(1):11–24. 2. Martins RG, Parvathaneni U, Bauman JE, et al. Cisplatin and radiotherapy with or without erlotinib in locally advanced squamous cell carcinoma of the head and neck: a randomized phase II trial. J Clin Oncol. 2013;31(11):1415–1421. 3. Ang KK, Zhang Q, Rosenthal DI, et al. Randomized Phase III Trial of Concurrent Accelerated Radiation Plus Cisplatin With or Without Cetuximab for Stage III to IV Head and Neck Carcinoma: RTOG 0522. J Clin Oncol. 2014; In press. 4. Bradley J, Masters GA, Hu C, et al. An intergroup randomized phase III comparison of standard-dose (60 Gy) versus highdose (74 Gy) chemoradiotherapy (CRT) +/- cetuximab (cetux) for stage III non-small cell lung cancer (NSCLC): Results on cetux from RTOG 0617. In: 15th World Conference on Lung Cancer, 2013. Sydney, Australia; 2013. 5. Stephen AG, Esposito D, Bagni RK, McCormick F. Dragging ras back in the ring. Cancer Cell. 2014;25(3):272–281. 6. Eke I, Zscheppang K, Dickreuter E, et al. Simultaneous β1 integrin-EGFR targeting and radiosensitization of human head and neck cancer. J Natl Cancer Inst. 2015;107(2):dju419 doi:10.1093/jnci/dju419. 7. Goodman SL, Picard M. Integrins as therapeutic targets. Trends Pharmacol Sci. 2012;33(7):405–412. 8. Lee BY, Timpson P, Horvath LG, Daly RJ. FAK Signaling in Human Cancer as a Target for Therapeutics. Pharmacol Ther. 2014; In press. 9. Seguin L, Kato S, Franovic A, et al. An integrin beta(3)-KRASRalB complex drives tumour stemness and resistance to EGFR inhibition. Nat Cell Biol. 2014;16(5):457–468. 10. Brizzi MF, Tarone G, Defilippi P. Extracellular matrix, integrins, and growth factors as tailors of the stem cell niche. Curr Opin Cell Biol. 2012;24(5):645–651. 11. Stransky N, Egloff AM, Tward AD, et al. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333(6046):1157–1160. 12. Wang M, Kern AM, Hulskotter M, et al. EGFR-mediated chromatin condensation protects KRAS-mutant cancer cells against ionizing radiation. Cancer Res. 2014;74(10):2825–2834. 13. McDermott U, Sharma SV, Dowell L, et al. Identification of genotype-correlated sensitivity to selective kinase inhibitors by using high-throughput tumor cell line profiling. Proc Natl Acad Sci U S A. 2007;104(50):19936–19941. 14. Krause M, Yaromina A, Eicheler W, Koch U, Baumann M. Cancer stem cells: targets and potential biomarkers for radiotherapy. Clin Cancer Res. 2011;17(23):7224–7229.
