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 (), and demonstrate the ability of panitumumab to enhance the growth-inhibitory effects of radiation (). To examine underlying mechanisms behind these observations, we demonstrate that panitumumab reduces clonogenic survival, and blocks radiation-induced EGFR signaling (). We also confirm that panitumumab augments radiation-induced apoptosis and DNA-damage (, ), and present evidence that inhibition of EGFR nuclear translocation after radiation may underlie these findings (). These observations are consistent with preclinical studies that established a favorable interaction between cetuximab and radiation 9–16
, 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 (), 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. 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
() 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 (). 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 , 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
(). 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 () 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 (). 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 34–36
. 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 (). 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 (), and that panitumumab augments the anti-tumor efficacy of radiation in xenograft models of NSCLC and HNSCC (). 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.