PDF file - 621K, Supplementary Figure S1. Treatment with Cetuximab inhibits EGF-mediated EGFR phosphorylation under 2D and 3D cell culture conditions in a cell line-dependent manner. Supplementary Figure S2. Induction of Cetuximab-mediated apoptosis in non-irradiated and irradiated 3D grown SCC cells. Supplementary Figure S3. EGFR inhibition with Cetuximab results in minor Caspase 3 cleavage. Supplementary Figure S4. Treatment with Cetuximab modulates tyrosine and serine protein phosphorylation. Supplementary Figure S5. JNK inhibition using siRNA or pharmacological inhibitor (SP600125) reduces cell survival of SCC cells. Supplementary Figure S6. JNK inhibition impairs Cetuximab-mediated c-Jun S63 phosphorylation. Supplementary Figure S7. JNK2 knockdown sensitizes UTSCC5 cells to Cetuximab-mediated radiosensitization. Supplementary Figure S8. EGFR and JIP-4 co-precipitate in untreated FaDu 3D cell cultures
ARTICLE ABSTRACT
EGF receptor (EGFR) promotes tumor growth as well as radio- and chemoresistance in various human malignancies including squamous cell carcinomas (SCC). In addition to deactivation of prosurvival signaling, cetuximab-mediated EGFR targeting might concomitantly induce self-attenuating signaling bypasses. Identification of such bypass mechanisms is key to improve the efficacy of targeted approaches. Here, we show great similarity of EGFR signaling and radiation survival in cetuximab-treated SCC cells grown in a more physiologic three-dimensional extracellular matrix and as tumor xenografts in contrast to conventional monolayer cell cultures. Using phosphoproteome arrays, we observed strong induction of JNK2 phosphorylation potentially resulting from cetuximab-inhibited EGFR through c-jun-NH2-kinase (JNK)-interacting protein-4 (JIP-4), which was identified using an immunoprecipitation-mass spectrometric approach. Inhibition of this signaling bypass by JIP-4 or JNK2 knockdown or pharmacologic JNK2 inhibition enhanced cetuximab efficacy and tumor cell radiosensitivity. Our findings add new facets to EGFR signaling and indicate signaling bypass possibilities of cancer cells to improve their survival on cetuximab treatment. By deactivation of cetuximab–self-attenuating JNK2-dependent signaling, the cytotoxicity, and radiosensitizing potential of cetuximab can be augmented. Cancer Res; 73(1); 297–306. ©2012 AACR.
