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. 2009 Jul;8(7):1751-60.
doi: 10.1158/1535-7163.MCT-09-0188. Epub 2009 Jul 7.

EGFRvIII and c-Met pathway inhibitors synergize against PTEN-null/EGFRvIII+ glioblastoma xenografts

Bachchu Lal  1 , C Rory Goodwin, Yingying Sang, Catherine A Foss, Kathrine Cornet, Sameena Muzamil, Martin G Pomper, Jin Kim, John Laterra
Affiliations

EGFRvIII and c-Met pathway inhibitors synergize against PTEN-null/EGFRvIII+ glioblastoma xenografts

Bachchu Lal et al. Mol Cancer Ther. 2009 Jul.

Abstract

Receptor tyrosine kinase (RTK) systems, such as hepatocyte growth factor (HGF) and its receptor c-Met, and epidermal growth factor receptor (EGFR), are responsible for the malignant progression of multiple solid tumors. Recent research shows that these RTK systems comodulate overlapping and dynamically adaptable oncogenic downstream signaling pathways. This study investigates how EGFRvIII, a constitutively active EGFR deletion mutant, alters tumor growth and signaling responses to RTK inhibition in PTEN-null/HGF(+)/c-Met(+) glioma xenografts. We show that a neutralizing anti-HGF monoclonal antibody (L2G7) potently inhibits tumor growth and the activation of Akt and mitogen-activated protein kinase (MAPK) in PTEN-null/HGF(+)/c-Met(+)/EGFRvIII(-) U87 glioma xenografts (U87wt). Isogenic EGFRvIII(+) U87 xenografts (U87-EGFRvIII), which grew five times more rapidly than U87-wt xenografts, were unresponsive to EGFRvIII inhibition by erlotinib and were only minimally responsive to anti-HGF monoclonal antibodies. EGFRvIII expression diminished the magnitude of Akt inhibition and completely prevented MAPK inhibition by L2G7. Despite the lack of response to L2G7 or erlotinib as single agents, their combination synergized to produce substantial antitumor effects (inhibited tumor cell proliferation, enhanced apoptosis, arrested tumor growth, prolonged animal survival), against subcutaneous and orthotopic U87-EGFRvIII xenografts. The dramatic response to combining HGF:c-Met and EGFRvIII pathway inhibitors in U87-EGFRvIII xenografts occurred in the absence of Akt and MAPK inhibition. These findings show that combining c-Met and EGFRvIII pathway inhibitors can generate potent antitumor effects in PTEN-null tumors. They also provide insights into how EGFRvIII and c-Met may alter signaling networks and reveal the potential limitations of certain biochemical biomarkers to predict the efficacy of RTK inhibition in genetically diverse cancers.

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Figures

Figure 1
Figure 1. Delivery of iodinated anti-HGF mAb L2G7 to orthotopic glioma xenografts
(A) [125I]L2G7 was administered at time zero via tail vein to mice bearing right hemispheric U87wt glioma xenografts. Animals were sacrificed at the indicated post-injection times and the percent-injected dose per gram (% ID/g) within tumor-bearing (ipsilateral) and contralateral brain hemispheres were determined. (B) SPECT-CT performed 48 hours after a single intravenous injection of [125I]L2G7 shows localized increase of radioactivity within the right hemispheric tumor. N=4, * P < 0.01, compared to determination in ipsilateral brain at same time point. Results shown are representative of three replicate experiments.
Figure 2
Figure 2. Systemic anti-HGF mAb inhibits growth of orthotopic PTEN-null/EGFRvIII- glioma xenografts in a dose dependent manner
Anti-HGF mAb L2G7 (0.625–10.0 mg/kg) or isotype control mAb 5G8 (10 mg/kg) was administered i.p. twice/week for five injections to mice bearing U87wt glioma xenografts. Treatment began 5 days after tumor cell implantation (PID 5). Animals were sacrificed on PID 23 and tumor volumes quantified (A). Hemotoxylin and eosin stained brain sections show representative tumor xenografts (B). N=5, * P < 0.001 compared to control.
Figure 3
Figure 3. Systemic anti-HGF mAb therapy inhibits Akt and MAPK activation in orthotopic PTEN-null/EGFRvIII- xenografts
Mice bearing pre-established orthotopic U87wt xenografts were treated with anti-HGF L2G7 or control mAb (i.p. 5 mg/kg) every 2–3 days for a total of three injections. Tumors were dissected from brain 24 hours after the last dose and tumor cell lysates subjected to quantitative dual wavelength infrared immunoblot analysis for the phosphorylated (P) and total (T) forms of Akt and MAPK as described in Materials and Methods. Representative bands of triplicate determinations are shown. N=5, * P < 0.05 compared to controls. Results shown are representative of three replicate experiments.
Figure 4
Figure 4. EGFRvIII expression alters tumor xenograft growth and signaling responses to anti-HGF therapy
(A) Immunoblot analysis of whole cell protein isolated from U87wt and U87-EGFRvIII cell lines (cultured for 24 h in 0.1% serum conditions) demonstrates increased levels of Akt and MAPK activation (phosphorylation) in cells expressing EGFRvIII. Phosphorylated Akt (or MAPK) relative to total Akt (or MAPK) were quantified by dual infrared immunofluorescence imaging as described in Materials and Methods. (B and C) Growth responses of subcutaneous U87wt and U87-EGFRvIII tumor xenografts (N=5) to control mAb 5G8 or anti-HGF mAb L2G7 (5 mg/kg i.p., every alternate day). Arrows indicate days of each mAb injection. U87wt and U87-EGFRvIII were matched at the time of treatment initiation. (D–G) Tumor lysates were obtained from U87wt (D and F) and U87-EGFRvIII (E and G) xenografts 24 hours following each of the three L2G7 injections and subjected to immunoblot analysis of phospho (P) and total (T) Akt and MAPK. Immunoblots (A, D–G) were quantified using dual wavelength infrared immunofluorescence imaging as described in Materials and Methods. Levels of phospho/total (P/T) Akt and MAPK are relative to control conditions that are normalized to 1.0. Representative bands of triplicate determinations are shown. * P < 0.05, ** P < 0.001 compared to controls. Results are representative of at least three replicate experiments.
Figure 5
Figure 5
HGF and EGFRvIII inhibitors synergize against tumor growth but not against Akt and MAPK activation in PTEN-null/EGFRvIII+ xenografts. Mice bearing subcutaneous U87-EGFRvIII s.c glioma xenografts were treated with either control mAb (5G8) or with anti-HGF L2G7 (5 mg/kg i.p. twice/week) +/− erlotinib (150mg/kg by oral gavage, 6 days/week) or PBS from post-implantation days (PID) 8–15. Arrows (A) indicate days of mAb therapy. Tumor growth responses (N=5) (A) and immunoblot analyses of tumor lysates (N=3) obtained on PID 18 for phospho/total EGFRvIII (B), Met (C), Akt (D), and MAPK (E) are shown. Immunoblots (B–E) were quantified using dual near-infrared immunofluorescence imaging as described in Materials and Methods. Representative bands of triplicate determinations are shown. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 compared to controls. Results are representative of four replicate experiments.
Figure 6
Figure 6
HGF and EGFRvIII inhibition cooperatively/synergistically inhibit tumor growth and tumor cell proliferation and increase apoptosis in orthotopic PTEN-null/EGFRvIII+ xenografts. (A) Mice bearing i.c. U87wt or U87-EGFRvIII xenografts were treated with either control (5G8) or anti-HGF (L2G7) mAb +/− erlotinib beginning on PID 5 using doses described in Figure 5. Tumor volumes (N=5) were determined from histological analyses of brains obtained on PID 30 (U87wt) or PID 16 (U87-EGFRvIII). (B) Mice bearing i.c. U87-EGFRvIII xenografts were treated as in (A) from PID 5–21. Anti-HGF + erlotinib therapy substantially improved animal survival in a cooperative and possibly synergistic fashion, (N=10, p<0.01). (C and D) Brains obtained as in (A) were immunohistochemically analyzed for tumor cell proliferation (Ki67) and apoptosis (anti-cleaved caspase 3) indices as described in Materials and Methods. # P < 0.05 compared to U87wt control; N=5, * P < 0.05, ** = P < 0.01, and *** = P < 0.001 compared to U87-EGFRvIII control. Shown are representative images of Ki-67 (C) and anti-cleaved caspase 3 (D) immunohistochemistry.
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