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

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

Simultaneous &beta;1 integrin-EGFR targeting and radiosensitization of human head and neck cancer.

BACKGROUND: Signaling from integrins and receptor tyrosine kinases (RTKs) contributes substantially to therapy resistance of malignant tumors. We investigated simultaneous &beta;1 integrin-epidermal growth factor receptor (EGFR) targeting plus radiotherapy in human head and neck squamous cell carcinomas (HNSCCs). METHODS: Ten HNSCC cell lines were grown in three-dimensional laminin-rich extracellular matrix cell cultures and two of them as tumor xenografts in nude mice (n = 12-16 per group). Targeting of &beta;1 integrin and EGFR with monoclonal inhibitory antibodies (AIIB2 and cetuximab, respectively) was combined with x-ray irradiation. Clonogenic survival, tumor growth, and tumor control (evaluated by Kaplan-Meier analysis), apoptosis, phosphoproteome (interactome, network betweeness centrality analysis), receptor expression (immunohistochemistry), and downstream signaling (western blotting) were assessed. Various mutants of the integrin signaling mediator focal adhesion kinase (FAK) were employed for mechanistic studies. All statistical tests were two-sided. RESULTS: Compared with &beta;1 integrin or EGFR single inhibition, combined &beta;1 integrin-EGFR targeting resulted in enhanced cytotoxicity and radiosensitization in eight out of 10 tested HNSCC cell lines, which responded with an FAK dephosphorylation after &beta;1 integrin inhibition. In vivo, simultaneous anti-&beta;1 integrin/anti-EGFR treatment and radiotherapy of UTSCC15 responder xenografts enabled better tumor control compared with anti-EGFR monotherapy and irradiation (hazard ratio [HR] = 6.9, 95% confidence interval [CI] = 1.6 to 30.9, P = .01), in contrast to the SAS nonresponder tumor model (HR = 0.9, 95% CI = 0.4 to 2.3, P = .83). Mechanistically, a protein complex consisting of FAK- and Erk1-mediated prosurvival signals for radiation resistance, which was effectively compromised by &beta;1 integrin and EGFR blocking. CONCLUSIONS: Concomitant targeting of &beta;1 integrin and EGFR seems a powerful and promising approach to overcome radioresistance of HNSCCs

Targeting of beta 1 integrins impairs DNA repair for radiosensitization of head and neck cancer cells

beta 1 Integrin-mediated cell-extracellular matrix interactions allow cancer cell survival and confer therapy resistance. It was shown that inhibition of beta 1 integrins sensitizes cells to radiotherapy. Here, we examined the impact of beta 1 integrin targeting on the repair of radiation-induced DNA double-strand breaks (DSBs). beta 1 Integrin inhibition was accomplished using the monoclonal antibody AIIB2 and experiments were performed in three-dimensional cell cultures and tumor xenografts of human head and neck squamous cell carcinoma (HNSCC) cell lines. AIIB2, X-ray irradiation, small interfering RNA-mediated knockdown and Olaparib treatment were performed and residual DSB number, protein and gene expression, non-homologous end joining (NHEJ) activity as well as clonogenic survival were determined. beta 1 Integrin targeting impaired repair of radiogenic DSB (gamma H2AX/53BP1, pDNA-PKcs T2609 foci) in vitro and in vivo and reduced the protein expression of Ku70, Rad50 and Nbs1. Further, we identified Ku70, Ku80 and DNA-PKcs but not poly(ADP-ribose) polymerase (PARP)-1 to reside in the beta 1 integrin pathway. Intriguingly, combined inhibition of beta 1 integrin and PARP using Olaparib was significantly more effective than either treatment alone in non-irradiated and irradiated HNSCC cells. Here, we support beta 1 integrins as potential cancer targets and highlight a regulatory role for beta 1 integrins in the repair of radiogenic DNA damage via classical NHEJ. Further, the data suggest combined targeting of beta 1 integrin and PARP as promising approach for radiosensitization of HNSCC