Radiosensitization of epidermal growth factor receptor/HER2-positive pancreatic cancer is mediated by inhibition of Akt independent of ras mutational status

Abstract

Purpose: Epidermal growth factor receptor (EGFR) family members (e.g., EGFR, HER2, HER3, and HER4) are commonly overexpressed in pancreatic cancer. We investigated the effects of inhibition of EGFR/HER2 signaling on pancreatic cancer to elucidate the role(s) of EGFR/HER2 in radiosensitization and to provide evidence in support of further clinical investigations.

Experimental design: Expression of EGFR family members in pancreatic cancer lines was assessed by quantitative reverse transcription-PCR. Cell growth inhibition was determined by MTS assay. The effects of inhibition of EGFR family receptors and downstream signaling pathways on in vitro radiosensitivity were evaluated using clonogenic assays. Growth delay was used to evaluate the effects of nelfinavir on in vivo tumor radiosensitivity.

Results: Lapatinib inhibited cell growth in four pancreatic cancer cell lines, but radiosensitized only wild-type K-ras-expressing T3M4 cells. Akt activation was blocked in a wild-type K-ras cell line, whereas constitutive phosphorylation of Akt and extracellular signal-regulated kinase (ERK) was seen in lines expressing mutant K-ras. Overexpression of constitutively active K-ras (G12V) abrogated lapatinib-mediated inhibition of both Akt phosphorylation and radiosensitization. Inhibition of MAP/ERK kinase/ERK signaling with U0126 had no effect on radiosensitization, whereas inhibition of activated Akt with LY294002 (enhancement ratio, 1.2-1.8) or nelfinavir (enhancement ratio, 1.2-1.4) radiosensitized cells regardless of K-ras mutation status. Oral nelfinavir administration to mice bearing mutant K-ras-containing Capan-2 xenografts resulted in a greater than additive increase in radiation-mediated tumor growth delay (synergy assessment ratio of 1.5).

Conclusions: Inhibition of EGFR/HER2 enhances radiosensitivity in wild-type K-ras pancreatic cancer. Nelfinavir, and other phosphoinositide 3-kinase/Akt inhibitors, are effective pancreatic radiosensitizers regardless of K-ras mutation status.

FIGURE 1. Lapatinib inhibits EGFR and HER2 but does not radiosensitize pancreatic cancer cells

A, Lapatinib inhibits EGF-induced activation of EGFR and HER2 in all four cell lines. Cells were serum-starved and treated with lapatinib and/or EGF. Lysates were subjected to immunoprecipitation with anti-EGFR or anti-HER2 antibodies and then immunoblotted with an anti-phosphotyrosine antibody. B, T3M4 cells harvoring wild-type K-ras were radiosensitized by lapatinib treatment (p<0.0001) whereas lines harboring mutant K-ras (MIA PaCa-2, Capan-2, and PANC-1) were not radiosensitized by lapatinib. Plating efficiency of unirradiated cells in the presence or absence of lapatinib was not significantly different for any cell line. C, Lapatinib strongly inhibits constitutive phosphorylation of Akt in T3M4 cells harboring wild-type K-ras whereas cells harboring K-ras mutations (Capan-2, MIA PaCa-2 and PANC-1) show little/no inhibition of Akt phosphorylation. Cells were treated with lapatinib at the indicated doses for 2 hours and protein lysates collected and analyzed by western blot analysis with indicated antibodies.

FIGURE 2. Ectopic expression of mutant K-ras abrogates lapatinib-mediated radiosensitization of T3M4 cells

A, T3M4 cells were transfected with either empty vector (pCGN) or pCGN-K-ras(G12V)-HA, subjected to treatment with lapatinib (5 μM) or DMSO vehicle for 2 hours and protein lysates harvested and analyzed by western blot analysis with antibodies against p-ERK1/2, ERK1/2, p-Akt or Akt. Expression of K-ras(G12V) blocked lapatinib-mediated Akt inhibition in T3M4 cells. B, Clonogenic survival assays of T3M4 cells transfected with pCGN vector control showed radiosensitization by lapatinib (p<0.0001) whereas cells expressing pCGN-K-ras(G12V)-HA were completely resistant to the radiosensitizing effect of lapatinib treatment. Plating efficiency of unirradiated cells in the presence or absence of lapatinib was not significantly different for any cell line.

FIGURE 3. Pancreatic cancer cells are radiosensitized by inhibition of PI3K/Akt

A, Akt phosphorylation is inhibited by treatment with LY294002 (5, 10, or 20 μM) but not DMSO control for 2 hours prior to collection of protein lysates and analysis by western blot. B, In clonogenic survival assays, LY294002 (10 μM) radiosensitized all pancreatic cancer cell lines tested, regardless of K-ras mutation status (p ≤ 0.002 for each). Plating efficiency of unirradiated cells in the presence or absence of LY294002 was not significantly different for any cell line.

FIGURE 4. Nelfinavir inhibits Akt phosphorylation and radiosensitizes both wild-type and mutant K-ras pancreatic cell lines

A, Cells were treated with nelfinavir (1 μM) for 4 or 28 h, and levels of p-ERK1/2 and p-Akt assessed by western blotting as above. After 28 hours, phosphorylation of Akt, but not ERK1/2, was decreased. B, All cell lines tested were radiosensitized following pretreatment with nelfinavir (1 μM) for 26 hours prior to and 2 hours after radiation.

FIGURE 5. Nelfinavir inhibits Akt phosphorylation and radiosensitizes Capan-2 xenografts

A, Capan-2 cells (5 × 10⁶) were injected into the flank of nude mice and allowed to grow until palpable, prior to initiating treatment with indicated doses of nelfinavir administered via gastric gavage once daily. Mice were sacrificed on the 5^th day of therapy and cellular lysates analyzed by western blot for total-Akt, phospho-Akt, and α-tubulin. B, Mean tumor volumes (n=6 per group). C, Fractional product values showing synergy for nelfinavir plus radiation treatment with an observed:expected ratio of 1.5 ± 0.27 at day 25. D, Cumulative surviving fraction (CSF) following multiple small doses of radiation alone (solid line) and radiation + nelfinavir (dotted line). Based on the surviving fraction after 2 Gy (SF_2Gy) for Capan-2 cells (Fig. 4B) treated with radiation alone (SF_2Gy = 0.85) or radiation + nelfinavir (SF_2Gy = 0.68), where CSF = SF_2Gy^{^(# of 2 Gy fractions)} and using the assumptions of no repopulation, reoxygenation, or redistribution and a constant SF_2Gy over time.