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Int J Radiat Oncol Biol Phys. Author manuscript; available in PMC Aug 25, 2010.
Published in final edited form as:
PMCID: PMC2927815
NIHMSID: NIHMS220555
Augmentation of radiation response by panitumumab in models of upper aerodigestive tract cancer
Tim J. Kruser, MD,1 Eric A. Armstrong, MS,1 Amol J. Ghia, MD,1 Shyhmin Huang, Ph.D,1 Deric L. Wheeler, Ph.D,1 Robert Radinsky, Ph.D,2 Daniel J. Freeman, Ph.D,2 and Paul M. Harari, MD1
1Department of Human Oncology, University of Wisconsin School of Medicine and Public Health
2Department of Oncology Research, Amgen Inc, Thousand Oaks, CA
Reprint requests: Paul M. Harari, Box 3684 Clinical Science Center-K4, 600 Highland Avenue, Madison, WI 53792. Phone: (608) 263-5009; Fax: (608) 262-6256; harari/at/humonc.wisc.edu
Purpose
In this report, we examine the interaction between panitumumab, a fully human anti-EGFR monoclonal antibody, and radiation in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cell lines and xenografts.
Methods and Materials
HNSCC lines UM-SCC-1 and SCC-1483 as well as the NSCLC line H226 were studied. Tumor xenografts in athymic nude mice were utilized to assess the in vivo activity of panitumumab alone and in combination with radiation. In vitro assays were performed to assess the impact of panitumumab on radiation-induced cell signaling, apoptosis, and DNA damage.
Results
Panitumumab increased radiosensitivity as measured by clonogenic survival assay. Radiation-induced EGFR phosphorylation and downstream signaling through MAPK and STAT3 was inhibited by panitumumab. Panitumumab augmented radiationinduced DNA damage by 1.2–1.6-fold in each of the cell lines studied as assessed by residual γ-H2AX foci after radiation. Radiation-induced apoptosis was increased 1.4–1.9-fold by panitumumab, as evidenced by Annexin V-FITC staining and flow cytometry. In vivo, combination therapy with panitumumab and radiation was superior to panitumumab or radiation alone in H226 xenografts (p=0.01) and showed a similar trend in SCC-1483 xenografts (p=0.08). These in vivo findings correlated with immunohistochemistry examination of PCNA; panitumumab with radiation markedly reduced PCNA staining in tumor xenografts.
Conclusions
These studies identify a favorable interaction when combining radiation and panitumumab in upper aerodigestive tract tumor models, both in vitro and in vivo. These data suggest that clinical investigations examining the combination of radiation and panitumumab in the treatment of epithelial tumors warrant further pursuit.
Keywords: Panitumumab, radiation, EGFR
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase, and member of the ErbB/HER family of receptors. EGFR is overexpressed or mutated in a majority of epithelial malignancies, including squamous cell carcinoma of the head and neck (HNSCC) and non-small cell lung cancer (NSCLC) 1. The widespread overexpression of this receptor in epithelial cancers has led to the development of a series of EGFR inhibitors, including the small molecule tyrosine kinase inhibitors (TKI) erlotinib and gefitinib and the anti-EGFR monoclonal antibodies (mAb) cetuximab and panitumumab. These targeted therapies have recently gained FDA approval in the treatment of patients with HNSCC, NSCLC, pancreatic, and colorectal cancer.
The interaction between the EGFR and ionizing radiation was initially explored in the early 1990’s 24. Investigators noted an increase in EGFR expression in various cell lines following radiation exposure 2, 3 as well as an increase in EGFR phosphorylation 5, 6, which leads to increased EGFR signaling and tumor cell proliferation. Indeed, an inverse relationship between EGFR expression and radiosensitivity was subsequently identified 7, 8. A series of studies on the radiosensitizing effects of the human-mouse chimeric mAb cetuximab (C225) established the capacity of EGFR inhibition to enhance the effects of radiation, both in vitro and in vivo, in various tumor model systems 916.
Radiation plays a central role in the treatment of locally advanced HNSCC and NSCLC. In 2006, an international phase III trial that combined the EGFR inhibitor mAb cetuximab with radiation in HNSCC patients demonstrated a 10% improvement in overall survival without significantly augmenting radiation-induced toxicities for patients receiving the EGFR inhibitor 17. This trial provided primary support for FDA registration of cetuximab for use in conjunction with radiation in patients with locoregionally advanced HNSCC. Based on promising preliminary Phase II results 18, 19, further clinical studies evaluating EGFR inhibitors in conjunction with cytotoxic chemotherapy and radiation in HNSCC and NSCLC are in progress.
Panitumumab is a fully human monoclonal IgG2 antibody that has been demonstrated to bind EGFR, block binding of EGF to the receptor, and inhibit receptor autophosphorylation. This results in inhibition of cellular proliferation and tumor growth, and induction of apoptosis 20, 21. A phase III trial utilizing panitumumab has demonstrated a significant improvement in progression-free survival in patients with chemo-refractory metastatic colorectal cancer 22, leading to FDA approval in this setting. In HNSCC, panitumumab is being investigated in combination with chemoradiotherapy. In a recently completed phase I trial studying the addition of panitumumab to carboplatin, paclitaxel, and intensity-modulated radiotherapy for locally advanced HNSCC, panitumumab did not appear to increase the rate of acute and late toxicities over that expected from chemoradiation alone 44. Additional studies utilizing panitumumab with chemoradiation following induction chemotherapy are ongoing. To date, the interaction between panitumumab and radiation has not been thoroughly investigated in pre-clinical models. The objective of the current study was to examine the interaction between panitumumab and radiation in xenograft models of HNSCC and NSCLC. In addition, we sought to explore mechanisms by which panitumumab modulates the antitumor efficacy of radiation.
Cell Lines
The human non-small cell lung cancer line NCI-H226 was obtained from the American Type Culture Collection (Rockville, MD) and maintained in complete culture media consisting of RPMI supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. Human HNSCC line UM-SCC-1 (floor of mouth) was provided by Dr. Thomas E. Carey (University of Michigan, Ann Arbor, MI) and SCC-1483 cells were provided by Dr. Jennifer Grandis (University of Pittsburgh, Pittsburgh, PA). SCC cells were cultured routinely in DMEM supplemented with 10% fetal bovine serum, 1 µg/ml hydrocortisone, and 1% penicillin and streptomycin. Cell culture media and supplements were obtained from Invitrogen (Carlsbad, CA).
Compounds and Antibodies
Panitumumab (ABX-EGF, Vectibix) was generously provided by Amgen Inc. (Thousand Oaks, CA). All other antibodies were purchased from commercial sources as indicated below: EGFR, pEGFR(Tyr1173), and HRP-conjugated goat-anti-rabbit IgG, goat-anti-mouse IgG and donkey-anti-goat IgG antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). PARP, Histone H3, pEGFR(Tyr845), PCNA, pSTAT3(Tyr705), and p-MAPK(Thr202/Tyr204) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Tubulin antibody was from Calbiochem (San Diego, CA). Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences Pharmingen (San Diego, CA). All other chemicals were purchased from Sigma (St. Louis, MO).
Subcellular fractionation and immunoblotting
Whole cell lysates were obtained using Tween-20 lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 20 mM beta glycerophosphate, and 10 µg/ml of leupeptin and aprotinin). For subcellular fractionation, cytoplasmic and nuclear extracts were prepared according to the instructions of the NE-PER® nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL). Protein was quantitated with a modified Bradford assay (Bio-Rad Laboratories, Hercules, CA) and equal amounts of protein were fractionated by SDS-PAGE. Thereafter, proteins were transferred to Immobilon-P membrane (Millipore, Billerica, MA) and analyzed by incubation with the appropriate primary antibody. Proteins were detected via incubation with HRP-conjugated secondary antibodies and enhanced chemiluminescence (ECL+) detection system (Amersham Biosciences, Piscataway, NJ). Where necessary protein bands were quantitated using ImageJ software (NIH, Bethesda, MD).
Clonogenic radiation survival
Survival after radiation exposure was defined as the ability of the cells to maintain their clonogenic capacity by forming colonies after radiation exposure. Cells were exposed to either panitumumab or human IgG for 72 h and subsequently irradiated at 0 to 8 Gy with a Shepherd & Associates Model 109 irradiator (San Fernando, CA) and a 137cesium hotbox source. Plates were rinsed with PBS/ 0.02 % EDTA, and cells were detached using 0.05% Trypsin/EDTA (Invitrogen, Carlsbad, CA), counted, and seeded for colony formation in six-well plates at 100 to 8,000 per well. Cells were incubated from 14 to 21 days with medium changes every 48 to 72 h. At the end of the experiment, colonies were stained with crystal violet in methanol and manually counted. Colonies consisting of 50 or more cells were scored, and 6 replicate wells containing 10 to 150 colonies per well were counted for each condition.
Assessment of apoptosis
Apoptosis was detected by flow cytometry via examination of altered plasma membrane assymetry using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit I (BD Biosciences, San Jose, CA). Phosphatidylserine (PS) is first exposed at the outer leaflet of the plasma membrane in the early stages of apoptosis and is detected by annexin V-FITC binding; combination with propidium iodide (PI) staining allows early and late apoptotic stages to be distinguished. Specifically, cells were treated with either panitumumab or control for 24 hours, and then exposed to radiation, 0–9 Gy. Forty-eight hours following irradiation, culture medium was collected (to retain detached cells), plates were rinsed with PBS/ 0.02 % EDTA, and cells were detached using 0.05% Trypsin/EDTA and combined with their medium and floating cells. Cells were washed twice in cold PBS and resuspended in the provided binding buffer at 106 cells per mL. Each sample was incubated with 5 uL each of the provided annexin V-FITC and PI solutions for 30' in the dark; volumes were increased to 500 uL and samples were run utilizing a BD FACScan flow cytometer using CellQuest software (Becton Dickinson, Franklin Lakes, NJ) for acquisition and analysis.
Quantification of γ-H2AX Foci
Cells were plated on chamber slides and exposed to 25 nM panitumumab or human IgG for one hour prior to irradiation. Cells were irradiated, fixed 24 hours after irradiation in 2% paraformaledhyde/3% sucrose solution for 10 minutes, then permeabilized for 5 minutes in 20 mM HEPES pH7.4, 50 mM NaCl, 3 mM MgCl2, 300mM sucrose, 0.5% Triton X-100. Cells were incubated with anti-phospho-γ-H2AX antibody (Upstate, Billerica, MA) dissolved in 2% BSA Fraction V 1:800 for 20 minutes. Cells were then incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG dissolved in 2% BSA Fraction V 1:100 for 20 minutes, and cells were then mounted using ProLong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA). Fluorescence images were captured using an Olympus BX51 Epifluorescent microscope fitted with a SPOT RT color camera and analyzed with the SPOT Advanced software (Diagnostic Instruments, Sterling Heights, MI). Visual scoring of at least 100 random cells per treatment condition was performed manually.
PARP Cleavage Analysis
Plates containing exponentially growing cells were exposed to either panitumumab (25 nM) or human IgG for 6 hours and subsequently irradiated with 0 to 9 Gy. Forty-eight hours after irradiation the media was collected to retain detached cells, centrifuged at 4°C, and cell pellets resuspeded in Tween-20 lysis buffer, as described above. The suspension was then added back to the appropriate PBS-rinsed plate in order to lyse remaining adherent cells. Immunoblotting was performed as described above utilizing a PARP antibody (Cell Signaling Technology, Beverly MA) to detect endogenous levels of full length PARP1 (116 kDa), as well as the large fragment (89 kDa) of PARP1 resulting from caspase cleavage.
Tumor Growth in Athymic Nude Mice
Athymic nude mice (3–4-week-old males) were obtained from Harlan Bioproducts for Science (Indianapolis, IN), maintained in microisolator cages and handled in laminar air-flow cabinets under aseptic conditions. The care and treatment of experimental animals was in accordance with institutional guidelines. Cells (~2 × 106) from the respective human cancer lines were injected subcutaneously into the flank area on day 0. Tumor volume was determined by direct measurement with calipers and calculated by the formula (π)/6 × (large diameter) × (small diameter)2. Panitumumab and control IgG were administered by intraperitoneal injection at specified doses twice weekly. Radiation treatment was delivered via a Philips RT-250 orthovoltage unit (Philips Medical Systems, Bothell, WA) using custom-designed mouse jigs. These jigs immobilized the animals and specifically exposed the dorsal flank (harboring tumor xenografts) for irradiation while minimizing exposure of non-tumor-bearing normal tissues.
Immunohistochemistry
The expression of proliferating cell nuclear antigen (PCNA) was detected in histologic sections of tumor xenografts. Briefly, excised tumor specimens were fixed in 10% neutral buffered formalin. Following embedding in paraffin, 5-µm sections were cut, and tissue sections were mounted. Sections were dried, deparaffinized, and rehydrated. Antigen unmasking was performed by heat-induced epitope retrieval with 10 mM sodium citrate buffer, pH 6.0 in a steamer for 20 minutes. After quenching endogenous peroxidase activity and blocking nonspecific binding sites, slides were incubated at 4°C overnight with 1:4000 dilution of primary antibody directed against PCNA, followed by a 30-min incubation of secondary antibody. Slides then were incubated with streptavidin peroxidase, visualized using the 3,3'-diaminobenzidine chromogen (Lab Vision Corp, Fremont, CA), dehydrated, and mounted.
Panitumumab-induced tumor growth inhibition
Antitumor activity of panitumumab has been demonstrated in pancreatic, renal, breast, prostate, ovarian, and colon tumor xenografts expressing various levels of EGFR 20, 21. To assess the antitumor efficacy in xenograft models of HNSCC and NSCLC, the dorsal flanks of athymic nude mice were subcutaneously inoculated with UMSCC-1, SCC-1483, and H226 tumor cells. Subsequently, mice bearing established xenografts were treated with increasing doses of panitumumab, administered via intraperitoneal injection twice weekly. As shown (Figure 1), panitumumab exhibited dose-dependent growth inhibition of tumors in each of the xenograft models. After 4 weeks of treatment, 10 ug doses of panitumumab induced a 1.6-fold reduction in volume in H226 tumors (p=0.05) and a 3.1-fold reduction in UM-SCC1 tumor volumes (p=0.01). In SCC-1483 tumors, 3 weeks of 25 ug doses of panitumumab induced a 1.9-fold reduction in tumor volumes (p=0.02). These respective drug doses in the less sensitive cell lines (SCC-1483 and H226) were chosen for subsequent in vivo studies with radiation. Weight measurements were collected weekly (data not shown), and no weight loss or other discernible toxicity was noted in mice receiving panitumumab.
Figure 1
Figure 1
In vivo efficacy of panitumumab (PNT) in HNSCC (A, B) and NSCLC (C) xenograft models. 2 × 106 cells were inoculated into the flank of aythmic mice (Day 0) as described in Materials and Methods. Mice were treated with indicated doses of PNT twice (more ...)
Panitumumab modulates in vitro radiation response
To examine the interaction of panitumumab with radiation in human tumor cells we conducted experiments assessing the impact of panitumumab on clonogenic survival following radiation. Tumor cells were exposed to panitumumab (25 nM) for 72 hours prior to irradiation. As shown in Figure 2, panitumumab pre-treatment induced modest but consistent radiosensitization in UM-SCC1 at 2, 4, 6, and 8 Gy (p < 0.01) and in H226 at 4, 6, and 8 Gy (p < 0.02). No significant radiosensitization was observed in the SCC-1483 cells.
Figure 2
Figure 2
Impact of panitumumab (PNT) on radiation response. Clonogenic survival was assessed in H226 (A) and UM-SCC1 (B) cells after exposure to radiation as described in Materials and Methods. Cells were exposed to either panitumumab (25 nM) or non-specific IgG (more ...)
Panitumumab inhibits radiation-induced EGFR activation
To assess the impact of radiation and panitumumab on EGFR activation and signaling pathways we examined whole cell lysates from irradiated UMSCC-1 cells that were pretreated with panitumumab. Irradiated cells without panitumumab pretreatment demonstrated a dose-responsive augmentation of EGFR phosphorylation at Tyrosine sites 845 and 1173 (Figure 2). Furthermore, it appears that the increased activation of EGFR after irradiation can increase downstream signaling as demonstrated by an enhancement of the phosphorylated version of STAT3, and that preadministration of panitumumab is able to inhibit radiation-induced EGFR signaling.
Panitumumab augments radiation-induced apoptosis
We employed two methods to examine whether the blockade of EGFR by panitumumab enhanced radiation-induced apoptosis in HNSCC and NSCLC cell lines. Flow cytometric analysis was performed on SCC-1483 and H226 cells utilizing Annexin V-FITC staining after radiation and demonstrated a dose-dependent induction of apoptosis by radiation treatment (Figure 3). Panitumumab alone caused a mild increase in the percentage of cells undergoing early apoptosis (high Annexin V-FITC, low PI staining) in SCC-1483 and H226 cells. Panitumumab pretreatment also augmented the impact of radiation on induction of apoptosis. At doses from 3 to 9 Gy, panitumumab increased the percentage of cells undergoing early apoptosis by 1.7- to 1.9-fold in H226 cells (p < 0.02). In SCC-1483 cells, at doses of 3 and 6 Gy panitumumab resulted in a 1.4- to 1.9-fold increase in cells undergoing apoptosis (p < 0.02). Furthermore, in UMSCC-1 cells we analyzed cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), which is indicative of cellular commitment to a pathway of programmed cell death. Radiation doses of 3, 6, and 9 Gy caused modest PARP cleavage (Figure 3C), while pretreatment with panitumumab increased the level of cleaved PARP at each radiation dose.
Figure 3
Figure 3
Panitumumab (PNT) augments radiation-induced apoptosis. H226 (A) and SCC-1483 cells (B) were either treated with 25 nM PNT or IgG control 24 hours before radiation. 48 hours after radiation cells were stained with Annexin V-FITC and analyzed as described (more ...)
Panitumumab augments radiation-induced DNA damage
We hypothesized that blockade of EGFR signaling by panitumumab may impair post-radiation DNA damage repair, and may represent a mechanism by which panitumumab modulates clonogenic survival and apoptosis following radiation. To examine this possibility, we performed immunostaining for γ-H2AX foci to determine the effect of panitumumab on radiation-induced DNA double-strand breaks. Histone H2AX is phosphorylated in response to DNA double-strand breaks 23, and detection can be used as a surrogate marker for DNA damage 24. UM-SCC1, SCC-1483, and H226 cells were exposed to increasing doses of radiation in the presence of either panitumumab or IgG control, and cells were stained for residual foci 24 hours after radiation. While panitumumab alone had minimal effect on DNA damage, pretreatment with panitumumab significantly increased the number of residual foci in SCC-1483 cells by a factor of 1.3 to 1.5 after 2, 4, and 6 Gy of radiation (p < 0.03) (Figure 4). A similar impact of panitumumab in combination with radiation on residual γ-H2AX foci (1.2- to 1.6- fold increases) were seen in SCC-1 cells at 2 and 4 Gy (p <0.05) and in H226 cells at 4 and 6 Gy (p < 0.01).
Figure 4
Figure 4
Panitumumab augments radiation-induced DNA damage. Cells from HNSCC lines UM-SCC1 (A) and SCC-1483 (B) and NSCLC line H226 (C) were exposed to either human IgG or panitumumab (PNT, 25 nM) for 1 hour before radiation. Cells were then incubated for 24 hours (more ...)
Panitumumab abrogates EGFR nuclear translocation after radiation
The relationship between nuclear translocation of the EGFR following radiation exposure has been well-studied 25. Following irradiation, EGFR is known to move to the nucleus and subsequently enhance the kinase activity of nuclear DNA-dependent kinase, a key enzyme responsible for repair of DNA-double strand breaks. To examine the ability of panitumumab to impact this nuclear translocation of EGFR following radiation, we separated nuclear and cytosolic fractions of cell lysate following irradiation of UMSCC-1 cells. Figure 5 demonstrates that the fraction of cellular EGFR in the nucleus is increased 20 minutes after irradiation, and that pretreatment with panitumumab limits this radiation-induced nuclear translocation.
Figure 5
Figure 5
Panitumumab blocks radiation-induced EGFR nuclear translocation in SCC-1 cells. Panitumumab (PNT, 25 nM) or non-specific IgG was adminstered 1 hour before radiation (3 Gy). (A) Cells were lysed 20 minutes after radiation and cytosolic and nuclear fractions (more ...)
Panitumumab augments in vivo radiation response
To examine the effects of combining panitumumab and radiation in vivo we inoculated the dorsal flanks of athymic mice with SCC-1483 and H226 tumor cells. Tumors were allowed to grow to approximately 200 mm3, and then randomized into 4 groups (n=10 per group in each experiment). Mice bearing established xenografts were subsequently treated with control IgG, radiation alone, panitumumab alone, or panitumumab and radiation in combination. To demonstrate the in vivo interaction of panitumumab and radiation, low doses of both agents were selected so that independent effects on tumor growth would be modest. Following three weeks of treatment, H226 xenografts treated with radiation alone were 2.5-fold smaller than control tumors (p=0.01), while the H226 xenografts treated with combined radiation and panitumumab were 4.7-fold smaller than control (p=0.001). The difference between radiation alone and combined radiation and panitumumab arm was significant (p =0.01) (Figure 6). Three weeks following treatment initiation SCC-1483 xenografts treated with panitumumab alone were 2.4-fold smaller than control tumors (p=0.06) and xenografts treated with radiation alone were 2.6-fold smaller than control tumors (p=0.09). The tumors treated with combination therapy were 11.9-fold smaller than control tumors (p=0.003). Combination therapy also showed significant growth inhibition compared with panitumumab alone (p=0.02), and a trend compared with radiation alone (p=0.08).
Figure 6
Figure 6
In vivo activity of panitumumab (PNT) +/− radiation in tumor xenografts. (A) SCC-1483 (2 × 106) or (B) H226 (2×106) cells were inoculated subcutaneously into the flank of athymic mice as described in Materials and Methods. Mice (more ...)
To further characterize the in vivo interaction between panitumumab and radiation, we assessed markers of tumor proliferation by immunohistochemistry (IHC). Twenty-four hours following the last tumor measurement and administration of treatment, xenografts were harvested from each treatment group and fixed in 10% formalin. Examination of proliferating cell nuclear antigen (PCNA) by IHC revealed large percentages of cells with dark, positive nuclear staining for PCNA in control tumors in both SCC-1483 and H226 xenografts (Figure 6). Panitumumab alone had minimal effect on PCNA staining intensity, while radiation alone did have a modest effect on the expression of PCNA. The combination of panitumumab and radiation markedly reduced the fraction of cells with detectable PCNA, and the intensity of PCNA staining. These results suggest that panitumumab may augment antitumor activity of radiation in HNSCC and NSCLC by enhancing the anti-proliferative effect of radiation.
This study presents evidence that the fully human EGFR mAb panitumumab augments radiation efficacy in models of upper aerodigestive tract cancer. We identify consistent in vivo activity of panitumumab in xenograft models of HNSCC and NSCLC (Figure 1), and demonstrate the ability of panitumumab to enhance the growth-inhibitory effects of radiation (Figure 6). To examine underlying mechanisms behind these observations, we demonstrate that panitumumab reduces clonogenic survival, and blocks radiation-induced EGFR signaling (Figure 2). We also confirm that panitumumab augments radiation-induced apoptosis and DNA-damage (Figures 3, ,4),4), and present evidence that inhibition of EGFR nuclear translocation after radiation may underlie these findings (Figure 5). These observations are consistent with preclinical studies that established a favorable interaction between cetuximab and radiation 916, and ultimately contributed to the clinical development of this agent in combination with radiation.
Strong evidence indicates that accelerated tumor cell proliferation contributes to treatment failure in HNSCC 26 and NSCLC 27. Radiation-induced activation of EGFR has been proposed as a mechanism contributing to accelerated cellular repopulation following radiotherapy 28. We show that panitumumab can prevent radiation-induced phosphorylation of EGFR and downstream signaling pathways (Figure 2), including activation of MAPK. To validate that antiproliferative effects accompany the combination of panitumumab with radiation in vivo, we analyzed tumor xenografts for PCNA expression by IHC. Figure 6 demonstrates that PCNA expression is reduced in xenografts treated with the combination of panitumumab and radiation compared to either modality alone. The anti-proliferative effects of panitumumab in vivo (Figure 1) and the ability of panitumumab to inhibit accelerated EGFR-dependent proliferative signaling after radiation may contribute to the observed augmentation of the antitumor efficacy of radiation in these models.
In addition to effects on proliferation, we investigated the impact of panitumumab on clonogenic survival after irradiation (Figure 2). We observed that panitumumab reduced clonogenic survival of UMSCC-1 and H226 cells. These results mirror the effect our group has previously shown in these cell lines using the EGFR TKI erlotinib 29. We hypothesized that panitumumab may enhance DNA damage after irradiation. As shown in Figure 4, panitumumab in combination with radiation enhanced residual γ-H2AX foci 24 hours after irradiation in all three cell lines. The magnitude of this effect is highly similar to the impact of cetuximab on DNA damage repair 30.
To further examine the interaction between panitumumab and radiation we examined apoptotic response. EGFR signaling is known to induce anti-apoptotic effects, and EGFR blockade has been shown to stimulate apoptosis 9. We demonstrate the ability of panitumumab to augment radiation-induced apoptosis in vitro (Figure 3). We have previously demonstrated enhanced apoptosis with various EGFR inhibitors and radiation combinations 9, 29, 31. Apoptosis occurs in response to DNA damage; therefore augmentation of radiation-induced DNA damage by panitumumab (Figure 4) may enhance the apoptotic response to irradiation.
The augmentation of radiation-induced apoptosis by panitumumab may also be mediated by blockade of radiation-induced EGFR-pSTAT3 signaling. We demonstrate that radiation can trigger phosphorylation of EGFR at tyrosine residues 1173 and 845 and increase levels of pSTAT3, and that panitumumab can block radiation-induced upregulation of this signaling pathway (Figure 2). The interaction between STAT3 and EGFR is well characterized in HNSCC 32 and NSCLC 33; phosphorylated Tyr 845 serves as a docking site for proteins such as STAT3, leading to STAT3 phosphorylation. Activated STAT3 plays a central role in protecting cells against apoptosis through the transcriptional modulation of survival genes, such as Bcl-xL, Bcl-2, and survivin 3436. Our findings are consistent with a report that STAT3 activation after either EGF stimulation or irradiation in breast cancer cells can be abolished by pretreatment with AG1478, an EGFR TKI 37.
Enhancement of radiation-induced DNA damage by panitumumab may represent a mechanism by which panitumumab reduces clonogenic survival and augments apoptosis after irradation. To explain the enhancement of radiation-induced DNA damage by panitumumab we examined the ability of panitumumab to block nuclear translocation of EGFR after irradiation. Dittman et al. have shown that radiation induces nuclear translocation of EGFR, which results in an increase in nuclear DNA-dependent protein kinase (DNA-PK) activity. EGFR blockade with the mAb cetuximab inhibits this process and enhances radiation-induced DNA damage 30, 38. In a similar fashion, we demonstrate an increase of EGFR in the nucleus of UM-SCC1 cells 20 minutes after radiation, and find that panitumumab can abrogate this shift of EGFR into the nucleus (Figure 5). Therefore, inhibition of nuclear translocation of EGFR by panitumumab may underlie enhancement of DNA damage in these cells lines, and contribute to enhanced apoptosis and radiosensitivity in the presence of EGFR blockade.
We demonstrate that panitumumab inhibits growth of HNSCC and NSCLC xenografts in a dose-dependent manner (Figure 1), and that panitumumab augments the anti-tumor efficacy of radiation in xenograft models of NSCLC and HNSCC (Figure 6). These findings are consistent with previous studies of cetuximab, which demonstrate that EGFR mAb blockade can augment radiation response in HNSCC and NSCLC xenografts 9, 14, 16. Although panitumumab augmented apoptosis in all 3 tumor cell lines tested, there was no clear impact on clonogenic survival in the SCC-1483 cells. Nevertheless, the combination of panitumumab and radiation demonstrated enhanced antitumor efficacy in SCC-1483 xenografts, suggesting that features beyond the in vitro environment may contribute to the anti-tumor effects. For example, inhibition of tumor angiogenesis by EGFR blockade would not be apparent in culture, but has been demonstrated to have in vivo importance 15, 31, 39. In addition, the magnitude of effects observed from single fraction studies in vitro may be compounded when tumor xenografts are exposed to multifraction regimens of radiation.
These preclinical results with panitumumab and radiation are promising and closely resemble findings from preclinical studies of cetuximab and radiation. Because of its fully human sequence, a potential comparative strength of panitumumab is the lower risk for immunogenicity and allergic reactions 40. On the other hand, cetuximab has a human IgG1 backbone and has been demonstrated to trigger antibody-dependent cellular cyotoxicity (ADCC) 41, 42. Clinical evidence suggests that this may impact the efficacy of cetuximab 43 but panitumumab has an IgG2 backbone, and thus would not be expected to induce ADCC. Ultimately, the therapeutic efficacies of distinct EGFR mAbs are best evaluated in the context of controlled clinical trials.
In conclusion, these studies demonstrate that the fully human anti-EGFR mAb panitumumab can augment radiation response in HNSCC and NSCLC model systems in vitro and in vivo. These results parallel preclinical studies of the anti-EGFR mAb cetuximab that demonstrated favorable interaction with radiation, and contributed to the phase III trial demonstrating a survival benefit for cetuximab plus radiation in patients with advanced HNSCC. These preclinical data suggest that systematic clinical investigations to examine combinations of radiation and panitumumab in cancer therapy are warranted.
Acknowledgments
TJK and AJG are supported by NIH T32 grants (CA009614-17 Physician Scientist Training in Cancer Medicine)
DLW is a recipient of an American Cancer Society Postdoctoral fellowship Work supported in part by NIH/NCI grant R01 CA 113448-01 to PMH
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