The combination of epidermal growth factor receptor inhibitors with gemcitabine and radiation in pancreatic cancer

Abstract

Purpose: Gemcitabine-radiotherapy is a standard treatment for locally advanced pancreatic cancer. Clinical data have shown that gemcitabine plus erlotinib is superior to gemcitabine alone for advanced pancreatic cancer. Therefore, we investigated the effects of the combination of epidermal growth factor receptor inhibitors with gemcitabine and radiation on a pancreatic cancer model.

Experimental design: EGFR signaling was analyzed by measuring phosphorylated EGFR (pEGFR(Y845, (Y1173)) and AKT (pAKT(S473)) protein levels in pancreatic cancer cell lines and tumors. The effects of scheduling on gemcitabine-mediated cytotoxicity and radiosensitization combined with erlotinib were determined by clonogenic survival. In vivo, the effects of cetuximab or erlotinib in combination with gemcitabine-radiation on the growth of BxPC-3 tumor xenografts were measured.

Results: We found in vitro that gemcitabine induced phosphorylation of EGFR at Y845 and Y1173 that was blocked by erlotinib. Treatment of BxPC-3 cells with gemcitabine before erlotinib enhanced gemcitabine-mediated cytotoxicity without abrogating radiosensitization. In vivo, cetuximab or erlotinib in combination with gemcitabine-radiation inhibited growth compared with gemcitabine-radiation (time to tumor doubling: gemcitabine + radiation, 19 +/- 3 days; cetuximab + gemcitabine + radiation, 30 +/- 3 days; P < 0.05, erlotinib + gemcitabine + radiation 28 +/- 3 days; P < 0.1). Cetuximab or erlotinib in combination with gemcitabine-radiation resulted in significant inhibition of pEGFR(Y1173) and pAKT(S473) early in treatment, and pEGFR(Y845), pEGFR(Y1173), and pAKT(S473) by the end of treatment. This study shows a novel difference pEGFR(Y845) and pEGFR(Y1173) in response to EGFR inhibition.

Conclusions: These results show that the EGFR inhibitors cetuximab and erlotinib increase the efficacy of gemcitabine-radiation. This work supports the integration of EGFR inhibitors with gemcitabine-radiation in clinical trials for pancreatic cancer.

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 24 hours with 3uM erlotinib beginning 24 hours after gemcitabine exposure. Cells were harvested for immunoblotting at 24 and 48 hours post-gemcitabine (t_{24, 48}) or immediately after erlotinib (t24, 48). The amounts of the indicated proteins are shown from a single experiment for each cell line that is representative of at least 3 independent experiments.

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 was calculated as the fraction of surviving colonies in treated cells relative to the number of colonies in the untreated control cells (surviving fraction = 1) (B). The radiation survival curves in response to gemcitabine and/or erlotinib (C) were used to calculate the mean inactivation dose (MID). Radiation enhancement (ER) was calculated as the ratio of the MID for drug treated cells to non-drug treated cells (ER = 1). Data are from the mean of 3 independent experiments ± standard error (B, D) or a single experiment (C). Statistically significant differences between gemcitabine versus erlotinib plus gemcitabine (B) or radiation versus radiation plus gemcitabine and/or erlotinib (D) are shown (*P<0.05).

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), or radiation (1 Gy × 10) as illustrated (A). Data are expressed as the time to tumor volume doubling in response to treatments that began on day 0 (B). The tic marks represent the mean time to tumor volume doubling and the vertical bars represent the 95% confidence intervals. Data are from 2 tumors/mouse with 6-7 mice per treatment group. Statistically significant differences are indicated in Table 1.

Figure 4

The effects of cetuximab or erlotinib, gemcitabine, and radiation on EGFR signaling in vivo. Mice were treated as described in Figure 3. Tumors were harvested on day 2 (A-D) or 12 (E-H) of treatment for immunoblotting. Data are from a single experiment (A, E) or the mean of 2-5 tumors ± standard error (B-D, F-H). Statistically significant differences of control versus treated* or EGFR^(Y845) versus EGFR^(Y1173)‡ are indicated (P<0.05).