Combined beta1 integrin/EGFR targeting of head and neck cancers <em>in-vitro</em> and <em>in-vivo</em>.

Abstract

Tumor cell resistance to radiotherapy and chemotherapy including novel targeted drugs is a great challenge for cancer treatment. One resistance mechanism found in cancer cells is the activation of bypass signaling which reduces the efficacy of the applied therapy. Therefore combined targeting of multiple pathways might increase tumor radiosensitivity and cell death. In this study, we examined the effect of dual beta1 integrin/EGFR inhibition in combination with irradiation in 10 head and neck squamous cell carcinoma (HNSCC) cell lines using 3D cell cultures and xenograft models. &nbsp; 3D colony formation assays, Western blotting, sequential immunoprecipitation-mass spectrometry, immunofluorescence staining, phosphoproteome arrays (including Reactome-based bioinformatics), and proximity ligation assays were performed. Downregulation of specific proteins was achieved by siRNA technology. Additionally, wildtype and constitutively active forms of Focal Adhesion Kinase (FAK) tagged to GFP were stably expressed in HNSCC cells. With regard to beta1 integrin targeting, one responder HNSCC model (UTSCC15) and one non-responder model (SAS) were transplanted subcutaneously on the legs of nude mice and treated with monotherapy or combination therapy of AIIB2 (monoclonal anti-beta1 integrin antibody) and Cetuximab plus/minus radiotherapy (20 Gy single dose). Tumor control and growth delay as well as signaling were assessed. The level of significance was determined by unpaired, 2-sided Student&acute;s t-test and Mann-Whitney-U-test using Microsoft Excel 2003 or log-rank test (actuarial estimates for time to local tumor recurrence were obtained using the Kaplan-Meier method). &nbsp; beta1 integrin targeting resulted in activation of Erk1/2 signaling pathway via FAK. We were able to prevent this activation by additional Cetuximab treatment, which enhanced both cytotoxicity and radiosensitization of AIIB2 monotherapy in-vitro and in-vivo. In-vitro, the ratio of responder to non-responder models for radiosensitization by beta1 integrin targeting was 8:2. Intriguingly and in contrast to SAS non-responder tumor xenografts, UTSCC15 responder tumor xenografts were completely controlled upon AIIB2/Cetuximab/radiotherapy. Mechanistically, we found a protein complex consisting of FAK and Erk1 that connects beta1 integrin and EGFR downstream signaling pathways. Targeting of beta1 integrin induced dissociation of this complex and hyperphosphorylation of the Ras-Raf-MEK-Erk signaling axis downstream of EGFR. To effectively prevent this bypass signaling, we simultaneously blocked beta1 integrin and EGFR, which could be causatively linked to a significantly higher radiosensitization as compared to monotherapies. Reactome bioinformatics clearly revealed a higher number of deactivated signaling hubs upon combined treatment relative to single treatments. As FAK seemed central to beta1 integrin and EGFR signal transduction, we used our FAK transfectants and found that the constitutively active form of FAK decreased the cellular radiosensitivity, abrogated HNSCC cell susceptibility to both AIIB2 and Cetuximab, and was downstream of Erk1. &nbsp; In summary, our data provide evidence for the superiority of simultaneous beta1 integrin/EGFR targeting over single targeting with regard to cytotoxicity and radiosensitization of HNSCC. In general, inhibition of overexpressed transmembrane receptors enables the deactivation of large parts of the prosurvival signaling network and, thus, represents a promising approach for patients with HNSCC tumors