We began this study by examining the effect of gemcitabine and erlotinib on EGFR signaling in several pancreatic cancer cell lines. Since our previous work in head and neck cancer cells demonstrated that gemcitabine induced phosphorylation of EGFR at the Src-dependent phosphorylation site, Y845, we investigated this site as well as the autophosphorylation site, Y1173. We found in the BxPC-3, Panc-1, and MPanc-96 cell lines, treated for 2 hours with approximately IC30 (G1) or IC70 (G2) concentrations of gemcitabine that phosphorylation of EGFR was induced at Y845 by the 48 hours post-treatment time point (). Phosphorylation of EGFR at Y1173 occurred in BxPC-3 and Panc-1 cells in response to gemcitabine. Furthermore, we found that a 24-hour exposure to 3uM erlotinib dramatically reduced the basal levels of pEGFR(Y845) and pEGFR(Y1173), as well as blocked the induction of pEGFR in response to gemcitabine. We also examined the levels of phosphorylated AKT (pAKT(S473)). In BxPC-3 cells, treatment with erlotinib, gemcitabine, or the combination of both resulted in reduced levels of pAKT(S473) (). In contrast, in Panc-1 and MPanc-96 cells, pAKT(S473) levels were resistant to erlotinib or gemcitabine alone and were only reduced in response to the combination.
Figure 1 The effects of gemcitabine and erlotinib on EGFR in pancreatic cancer cells. BxPc-3, Panc-1, or MPanc-96 cells were treated for 2 hours with 100nM (G1), 300nM (G2) (BxPC-3), 1uM (G1) or 3uM (G2) (Panc-1 and MPanc-96) gemcitabine. Cells were treated for (more ...)
To understand the influence of EGFR inhibitors on gemcitabine-mediated radiosensitization, we first examined cytotoxicity in response to two different schedules of gemcitabine and erlotinib. In the first schedule, a 2-hour gemcitabine treatment was followed 24 hours later by a 72-hour erlotinib exposure (). Treatment under this condition resulted in an enhancement of gemcitabine-mediated cytotoxicity by erlotinib (). In the second schedule, erlotinib was given for 72 hours and followed by a 2-hour gemcitabine exposure and a 22-hour gap before plating for clonogenic survival. In contrast to the first schedule, the second schedule produced no enhanced gemcitabinecytotoxicity by erlotinib. To further understand the importance of scheduling EGFR inhibitors with gemcitabine and radiation, we performed radiosensitization experiments under these two treatment conditions, in which irradiation was performed 24 hours after gemcitabine treatment. For both schedules 1 and 2, gemcitabine alone produced significant radiosensitization (RER 1.9 ± 0.10) that was unaffected by the addition of erlotinib (RER 1.9 ± 0.04 and 1.7 ± 0.05, respectively) (). Furthermore, erlotinib alone did not produce radiosensitization under either schedule. While it would have been optimal if erlotinib had enhanced gemcitabine-mediated radiosenstiziation, the finding that erlotinib did not abrogate gemcitabine-mediated radiosensitization was an acceptable outcome, since our clinical goal for pancreatic cancer is to improve systemic therapy while maintaining or improving local radiosensitization.
Figure 2 The effects of erlotinib on cytotoxicity and radiosensitization in response to gemcitabine. BxPC-3 cells were treated for 2 hours with 100nM gemcitabine and/or for 72 hours with 3uM erlotinib according to schedules 1 or 2 as illustrated (A). Cytotoxicity (more ...)
EGFR inhibitors have been shown to inhibit angiogenesis, invasion, and proliferation in addition to promote apoptosis (24
). Therefore, we wanted to test the effects of EGFR inhibition, gemcitabine, and radiation in a murine pancreatic tumor xenograft model. Since previous data showed that cetuximab could enhance the inhibition of pancreatic tumor growth by gemcitabine-radiation (25
), we wished to compare the efficacy of cetuximab and erlotinib. We selected relatively low doses of gemcitabine (120mg/kg) and radiation (1Gy X 10) for these studies, which would permit us to detect further inhibition of tumor growth by the addition of cetuximab or erlotinib if it occurred. We administered cetuximab once per week or erlotinib daily (each for 2 cycles and 4 hours post irradiation; ) based on the substantially longer half-life of cetuximab (95 hours) (26
) relative to erlotinib (36 hours) (27
). Treatment with gemcitabine, cetuximab, erlotinib, or radiation alone did not produce any effect on tumor growth (, ). As anticipated, the addition of gemcitabine to radiation extended the time required for tumor volume doubling, although it did not reach statistical significance (p=0.12). Cetuximab in combination with radiation produced a significant delay in the time to tumor volume doubling compared to either untreated tumors (Δ=10.4±4.5 days, p<0.03) or tumors treated with cetuximab alone (Δ=9.7±4.5 days, p<0.04). The time to tumor volume doubling was also significantly longer after treatment with gemcitabine and erlotinib compared to control (Δ=11.7±4.5 days, p<0.02) or gemcitabine alone (Δ=12.2±4.5 days, p<0.01). The most effective regimen for inhibition of tumor growth as evidenced by the significantly increased time to tumor volume doubling was the triple combination of either cetuximab or erlotinib plus gemcitabine-radiation (30.1±3.3 days or 27.5±3.3 days, respectively). The addition of cetuximab or erlotinib to gemcitabine and radiation produced minimal weight loss () suggesting that these combinations did not produce marked normal tissue toxicity. Taken together, these results demonstrate that EGFR antagonists such as cetuximab or erlotinib can improve the anti-tumor efficacy of gemcitabine-radiation (without additional normal tissue toxicity) in a pancreatic cancer model.
Figure 3 The effects of cetuximab or erlotinib on gemcitabine-mediated radiosensitization in vivo. Athymic nude mice bearing subcutaneous BxPC-3 xenografts were treated with the indicated combinations of gemcitabine (120mg/kg), cetuximab (50mg/kg), erlotinib (100mg/kg), (more ...)
Tumor volume doubling (days)
Relative* animal weight during and after therapy.
To characterize the molecular phenotype of tumors treated with cetuximab or erlotinib and gemcitabine-radiation, we analyzed EGFR signaling in BxPC-3 tumor xenografts both at the beginning (day 2; -)) and end (day 12; -) of the 2 week treatment cycle. Consistent with our in vitro findings, we found that phosphorylation of EGFR at Y845 was significantly reduced by treatment with cetuximab or erlotinib by treatment day 2 (both p<0.01), and we observed a trend for gemcitabine (p=0.11) or radiation (p=0.20) alone to increase EGFR(Y845) although the combination of gemcitabine and radiation did not (p=0.18). Interestingly, at this early time point, neither cetuximab nor erlotinib inhibited EGFR(Y845) in the presence of gemcitabine-radiation, despite the result that these treatments produced the greatest tumor growth inhibition. We therefore investigated EGFR(Y1173) and found that cetuximab or erlotinib alone significantly reduced EGFR(Y1173) relative to control (both p<0.01). In addition, the combination of either cetuximab or erlotinib with gemcitabine-radiation significantly reduced EGFR(Y1173) at the 2 day time point relative to control (p<0.03 and p<0.02, respectively). There were significant differences between EGFR(Y845) and EGFR(Y1173) phosphorylation in tumors treated with the combination of either cetuximab (p<0.02) or erlotinib (p<0.010) with gemcitabine-radiation; both cetuximab and erlotinib in combination with gemcitabine-radiation produced significant inhibition of EGFR(Y1173) but not EGFR(Y845), suggesting that these two phosphorylation sites respond differently to these combinations. When we assessed tumors at the end of the treatment cycle (day 12), we found significant inhibition of both EGFR(Y845) and EGFR(Y1173) in response to the single agents, gemcitabine, cetuximab, erlotinib, or radiation (all p<0.01). More importantly, the combination of cetuximab or erlotinib with gemcitabine-radiation resulted in significant inhibition of EGFR(Y845) and EGFR(Y1173) (relative to control; both p<0.01) demonstrating efficient inhibition of EGFR phosphorylation under the conditions which produced the greatest reduction in tumor growth. Together, these results suggest that inhibition of pEGFR(Y1173) in early treatment or EGFR(Y845) and pEGFR(Y1173) in late treatment are qualitatively (but not quantitatively) associated with tumor growth inhibition
Figure 4 The effects of cetuximab or erlotinib, gemcitabine, and radiation on EGFR signaling in vivo. Mice were treated as described in . Tumors were harvested on day 2 (A-D) or 12 (E-H) of treatment for immunoblotting. Data are from a single experiment (more ...)
We also examined the effects of cetuximab or erlotinib with gemcitabine and radiation on a pro-survival molecule downstream of EGFR, pAKT(S473). We found that the amount of pAKT(S473) in tumors following treatment with gemcitabine, cetuximab, erlotinib, radiation, or gemcitabine plus radiation was significantly reduced by the second day of treatment (all p<0.01). Furthermore, in response to either cetuximab or erlotinib in combination with gemcitabine-radiation, pAKT(S473) was also significantly reduced (relative to control; both p<0.01). The levels of pAKT(S473) remained repressed through out the duration of the treatment as evidenced by repression of pAKT(S473) compared to control pAKT(S473) levels at day 12 (p<0.002). Together these data demonstrate that cetuximab or erlotinib, as well as gemcitabine and radiation inhibit pAKT(S473).