Afatinib and Temozolomide combination inhibits tumorigenesis by targeting EGFRvIII-cMet signaling in glioblastoma cells

Abstract

Background: Glioblastoma (GBM) is an aggressive brain tumor with universal recurrence and poor prognosis. The recurrence is largely driven by chemoradiation resistant cancer stem cells (CSCs). Epidermal growth factor receptor (EGFR) and its mutant EGFRvIII are amplified in ~ 60% and ~ 30% of GBM patients, respectively; however, therapies targeting EGFR have failed to improve disease outcome. EGFRvIII-mediated cross-activation of tyrosine kinase receptor, cMET, regulates GBM CSC maintenance and promote tumor recurrence. Here, we evaluated the efficacy of pan-EGFR inhibitor afatinib and Temozolomide (TMZ) combination on GBM in vitro and in vivo.

Methods: We analyzed the effect of afatinib and temozolomide (TMZ) combination on GBM cells U87MG and U251 engineered to express wild type (WT) EGFR, EGFRvIII or EGFRvIII dead kinase, CSCs isolated from U87 and U87EGFRvIII in vitro. The therapeutic utility of the drug combination was investigated on tumor growth and progression using intracranially injected U87EGFRvIII GBM xenografts.

Results: Afatinib and TMZ combination synergistically inhibited the proliferation, clonogenic survival, motility, invasion and induced senescence of GBM cells compared to monotherapy. Mechanistically, afatinib decreased U87EGFRvIII GBM cell proliferation and motility/invasion by inhibiting EGFRvIII/AKT, EGFRvIII/JAK2/STAT3, and focal adhesion kinase (FAK) signaling pathways respectively. Interestingly, afatinib specifically inhibited EGFRvIII-cMET crosstalk in CSCs, resulting in decreased expression of Nanog and Oct3/4, and in combination with TMZ significantly decreased their self-renewal property in vitro. More interestingly, afatinib and TMZ combination significantly decreased the xenograft growth and progression compared to single drug alone.

Conclusion: Our study demonstrated significant inhibition of GBM tumorigenicity, CSC maintenance in vitro, and delayed tumor growth and progression in vivo by combination of afatinib and TMZ. Our results warrant evaluation of this drug combination in EGFR and EGFRvIII amplified GBM patients.

Keywords: Afatinib; Cancer stem cells; Epidermal growth factor receptor; Glioblastoma; Temozolomide.

Fig. 1

Afatinib and TMZ combination differentially inhibit proliferation and clonogenic survival of EGFR and EGFRvIII expressing GBM cells. a, b EGFR and EGFRvIII expression differentially effect cell proliferation. a U87MG, U87EGFR WT, U87EGFRvIII, U87EGFRvIII DK, U251, U251EGFR WT (Tet-inducible system) and U251EGFRvIII (Tet-inducible system) cell lysates were analyzed for EGFR (full length), EGFRvIII and pEGFR (Tyr-1068). b The graph shows increased proliferation rate of U87EGFRvIII cells compared to U87MG, U87EGFR WT, and U87EGFRvIII DK cells. Tet - tetracycline. c, d U87MG, U87EGFR WT, U87EGFRvIII and U87EGFRvIII DK cells were seeded and treated as specified for 48 h. After washing with PBS, cells were allowed to grow for 12 days in 2% media. Colonies formed were fixed with methanol, stained with crystal violet solution, dissolved in 10% acetic acid and absorbance was measured at 570 nm. The graph shows the mean (± SD) percentage of colony formation. The experiment was repeated three times (*^$ P ≤ 0.05); * significant compared to control; $ significant compared to TMZ. e, f Afatinib and TMZ combination decreases the anchorage-independent growth of TMZ-resistant EGFRvIII GBM cells. U87MG, U87 EGFR WT, U87EGFRvIII and U87EGFRvIII DK cells (5 × 10³) were seeded with 0.25% agarose on the top of the 0.5% base agar. After overnight incubation, cells were incubated with TMZ (25 μM) or afatinib (1 μM) or combination of both for a month. The 0.1% crystal violet stained colonies were counted (n = 3). The graph shows mean (± SD) number of small (blue) and big colonies (orange). (* P ≤ 0.05); * number of small colonies, significant compared to control; ^# number of big colonies, significant compared to control

Fig. 2

Afatinib inhibits EGFR and EGFRvIII activation and augments G₂/M arrest with TMZ. a U87MG, U87EGFR WT and U87EGFRvIII DK cells were incubated with TMZ (25 μM), afatinib (1 μM) or combination of drugs for 48 h, and cell lysates were analyzed for pEGFR (Tyr-1068) by western blot analysis. β-actin serves as a loading control. b U87EGFRvIII cells were treated with TMZ, afatinib, erlotinib or combination of TMZ and afatinib and analyzed for pEGFR (Tyr-1068). c U251 and U251EGFRvIII cells were treated with afatinib, TMZ or combination for 48 h. Cell lysates were analyzed for pEGFR (Tyr-1068) and pAKT (Ser-473) by western blot analysis. d U87EGFRvIII cells were synchronized with double thymidine block and treated with TMZ (25 μM), afatinib (1 μM) or combination of drugs for 48 h. Cells were trypsinized, fixed with 70% ethanol, stained with Telford reagent and analyzed by flow cytometry. e The bar diagram shows the mean (± SD) percentage of distribution of the cells (*^$# P ≤ 0.05); * significant compared to control; $ significant compared to TMZ; # significant compared to afatinib (n = 3)

Fig. 3

Afatinib and TMZ combination decreases migration and invasion of U87MG and U87 EGFRvIII cells. a, b U87MG and U87EGFRvIII cells (2.5 × 10⁴) were incubated with TMZ (25 μM), afatinib (1 μM) or combination of drugs for 48 h. Non-migrated cells in the upper chamber were removed with a cotton swab, and the migrated cells were stained and counted. Representative images are shown (10X magnification). The bar graph shows the mean (± SD) percentage of migrated cells. Experiments were repeated three times and 5 random fields were chosen for quantification (*^$ P ≤ 0.05); * significant compared to control; $ significant compared to TMZ. c, d Afatinib alone or in combination with TMZ decreases the invasion of U87EGFRvIII cells. U87MG and U87 EGFRvIII cells (5.0 × 10⁴) were incubated with TMZ (25 μM), afatinib (1 μM) or combination of drugs for 48 h. Non-invaded cells in the upper chamber was removed with a cotton swab, and the invaded cells were stained and counted. The bar graph shows the mean (± SD) percentage of invaded U87MG and U87EGFRvIII cells. Experiments were repeated three times (*^$ P ≤ 0.05); *significant compared to control; $ significant compared to TMZ. e, f Afatinib inhibits EGFRvIII-mediated JAK2/STAT3 and FAK signaling. U87EGFRvIII cells were treated with TMZ (25 μM), afatinib (1 μM) or combination for 48 h, and lysates were analyzed for pEGFR (Tyr-1068), pJAK2 (Tyr-1007/1008), pSTAT3 (Tyr-705), pFAK (Tyr-925) and pAKT (Ser-473) by western blot analysis. g EGFRvIII kinase domain mediates FAK (Tyr-925) activation. U87, U87 EGFR WT, U87EGFRvIII and U87EGFRvIII DK cell lysates were analyzed for pFAK (Tyr-925), pFAK (Tyr-576/577) and pFAK (Tyr-397)

Fig. 4

Afatinib inhibits EGFRvIII-cMET signaling crosstalk in SP/CSCs cells. a, b Afatinib reduces TMZ-resistant U87EGFRvIII SP/CSCs. U87MG and U87EGFRvIII cells treated with either TMZ (25 μM), afatinib (1 μM) alone or combination for 48 h were trypsinized, stained with Hoechst 33342 (5 μg/ml) and analyzed for CSCs and NSP cells using flow cytometry. The bar diagram shows the mean (± SD) percentage of SP/CSCs from U87MG and U87EGFRvIII (n = 3). c, d Afatinib decreases U87EGFRvIII-mediated self-renewal properties of CSCs. U87MG and U87EGFRvIII cells treated with TMZ (25 μM), afatinib (1 μM), or combination for 48 h were plated (2 × 10³ cells/well in 100 μl of stem cell medium) on a 96-well ultra-low attachment plate. Neurosphere/tumor spheres formed after 10 days were quantified and photographed (X20 magnification). The graph shows the mean (± SD) number of tumor spheres formed by U87MG and U87EGFRvIII cells (n = 4). (*^$ P ≤ 0.05); * significant compared to control; $ significant compared to TMZ. e U87, U87EGFR WT, U87EGFRvIII and U87EGFRvIII DK cell lysates were analyzed for pcMET (Tyr-1234/1235) by western blot analysis. f U87EGFRvIII cells were treated with either TMZ (25 μM), afatinib (1 μM), or combination for 48 h and analyzed for pcMET (Tyr-1234/1235). g Expression of Nanog, Oct3/4 (self-renewal marker) and pcMET (Tyr-1234/1235) were analyzed in SP/CSCs and NSP cells by western blot analysis. h U87EGFRvIII SP cells were treated with TMZ (25 μM), afatinib (1 μM), or combination for 48 h and analyzed for pcMET (Tyr-1234/1235) and stemness markers Nanog and Oct3/4 by western blot analysis. i U87EGFRvIII cells were treated with varying concentrations of the cMET specific inhibitor, SU-11274 (1 – 20 μM) or afatinib (1 μM) for 48 h and lysates were analyzed for pcMET (Tyr-1234/1235), Nanog and Oct3/4

Fig. 5

Afatinib and TMZ combination reduces tumor burden in vivo. a, b U87EGFRvIII luciferase transfected cells (2 × 10⁴ in 2 μl of PBS) were intracranially injected into 4–6 week’s old mice using a stereotactic frame. After 5 days, mice were randomized into 4 groups and treated with vehicle (n = 8), TMZ (25 mg/kg BW) (n = 7), afatinib (10 mg/kg BW) (n = 8) or combination (n = 5) for 5 days a week by oral gavage. Animals treated with TMZ alone or in combination with afatinib survived longer and were sacrificed after 30 days. The tumor volume (total photon count) was measured using IVIS imaging on days 0, 7, 9, 11, 20 and 30. (^{$, #} P ≤ 0.03); $ significant compared to TMZ; # significant compared to vehicle control (c) Kaplan Meier survival curve analysis showing effect of control, afatinib, TMZ or combination on OS of EGFRvIII orthograft mice. d, e Confocal microscopy showing expression of (d) pEGFR (Tyr-1068) (red staining), Oct3/4 (green staining) and (e) pcMET (Tyr-1234/1235) (red) and Nanog (green) images in U87EGFRvIII tumor xenografts

Fig. 6

Possible mechanism of afatinib-TMZ combination therapy in GBM. TMZ targets the differentiated proliferating GBM cells but fails to eradicate slow-growing CSCs and result in tumor progression. Afatinib decreases proliferation of U87EGFRvIII cells by inhibiting EGFRvIII/AKT signaling, cell migration and invasion by inhibiting EGFRvIII/JAK2/STAT and FAK (Tyr-925) signaling pathways. Afatinib by inhibiting EGFRvIII-cMET cross-activation decreases CSC stemness possibly by downregulating stemness transcription factors Oct3/4 and Nanog. Dotted line indicates possible effects