Inhibition of the growth of patient-derived pancreatic cancer xenografts with the MEK inhibitor trametinib is augmented by combined treatment with the epidermal growth factor receptor/HER2 inhibitor lapatinib

Abstract

Mutations of the oncogene KRAS are important drivers of pancreatic cancer progression. Activation of epidermal growth factor receptor (EGFR) and human EGFR2 (HER2) is observed frequent in pancreatic adenocarcinomas. Because of co-activation of these two signaling pathways, we assessed the efficacy of inhibition of EGFR/HER2 receptors and the downstream KRAS effector, mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK) kinase 1 and 2 (MEK1/2), on pancreatic cancer proliferation in vitro and in a murine orthotopic xenograft model. Treatment of established and patient-derived pancreatic cancer cell lines with the MEK1/2 inhibitor trametinib (GSK1120212) inhibited proliferation, and addition of the EGFR/HER2 inhibitor lapatinib enhanced the inhibition elicited by trametinib in three of eight cell lines. Importantly, in the orthotopic xenograft model, treatment with lapatinib and trametinib resulted in significantly enhanced inhibition of tumor growth relative to trametinib treatment alone in four of five patient-derived tumors tested and was, in all cases, significantly more effective in reducing the size of established tumors than treatment with lapatinib or trametinib alone. Acute treatment of established tumors with trametinib resulted in an increase in AKT2 phosphorylation that was blunted in mice treated with both trametinib and lapatinib. These data indicate that inhibition of the EGFR family receptor signaling may contribute to the effectiveness of MEK1/2 inhibition of tumor growth possibly through the inhibition of feedback activation of receptor tyrosine kinases in response to inhibition of the RAS-RAF-MEK-ERK pathway. These studies provide a rationale for assessing the co-inhibition of these pathways in the treatment of pancreatic cancer patients.

Figure 1

(A) Relative activation of selected RTKs for 15 patient-derived pancreatic cancer lysates and four established pancreatic cancer cell lines (black, more than three times the threshold; dark gray, two to three times the threshold; light gray, one to two times the threshold). (B) Western blots for EGFR and HER2 in four patient-derived pancreatic cancers, established pancreatic cancer cell lines, and SK-BR-3, a breast cancer cell line (asterisk represents a 1-s exposure).

Figure 2

Proliferation assays for four established pancreatic cancer cell lines (A–D) and four patient-derived pancreatic cancer cell lines (E–H) after treatment with DMSO (control), lapatinib (1.0 µM), trametinib (0.3 µM), or combination of lapatinib and trametinib. P values are given as follows: *P < .05, **P < .01, ***P < .001.

Figure 3

(A–C) Treatment of three patient-derived pancreatic cancer cell lines with DMSO (control) and lapatinib (1.0 µM), showing relative phosphorylation of EGF and FGF family RTKs (ND, no detection of pRTK). (D and E) Western blot analysis for ERK and pERK in three patient-derived pancreatic cancer cell lines and four established cell lines after treatment with DMSO, lapatinib (1.0 µM), trametinib (0.3 µM), and combination.

Figure 4

(A–E) In vivo response of five different human-derived pancreatic cancers after treatment with vehicle control, lapatinib (L; 65 mg/kg, twice daily), trametinib (0.3 mg/kg, daily), or combination (L + T). (F) Summary of liver metastasis for the same five tumors. (G) Observed liver metastases in mice bearing individual patient-derived tumors. P values are given as follows: *P < .05, **P < .01, ***P < .001.

Figure 5

Response of established tumors (A–C) following treatment with lapatinib (65 mg/kg, twice daily; circles), trametinib (0.3 mg/kg, daily; squares), or combination (triangles). The tumors were allowed to grow to 250 to 500 mm³ before the onset of treatment. Arrows denote a dose escalation of trametinib to 1.0 mg/kg daily. The right panels show MRIs at the specified time points for representative mice treated with the combination of lapatinib and trametinib with tumors outlined. P values are given as follows and indicate significance of combination treatment versus trametinib alone: *P < .05, **P < .01, ***P < .001.

Figure 6

In vivo effects of inhibitor therapy on MEK1/2 signaling. Mice orthotopically implanted with patient-derived tumors 366 (A), 608 (B), and 738 (C) were treated for 24 hours with vehicle control, lapatinib (65 mg/kg, twice daily), trametinib (3 mg/kg, daily), or combination of lapatinib (65 mg/kg) and trametinib (3 mg/kg, daily) and then sacrificed. Whole tumor lysates from these animals were analyzed by Western blot analysis with the indicated antibodies. Each lane represents the tumor lysate of an individual experimental mouse exposed to the indicated treatment condition.

Figure 7

Relative phosphorylation of 366 tumor (KRAS mut), 608 tumor (KRAS mut), and 738 tumor (KRAS wt) xenografts treated with vehicle control, lapatinib (65 mg/kg, orally, twice daily), trametinib (3 mg/kg, orally, daily), or combination of trametinib and lapatinib. (A–C) Histograms depicting relative pAkt1(S473), pAkt2(S474), pErk1(T202/Y204), p53(S46), and p70S6K(T421/S424) levels in treated 366 tumor (A), 608 tumor (B), and 738 tumor (C) xenografts. Significance is denoted as ***P < .001, **P < .01, and *P < .05.