Ellen Dickreuter

Simultaneous β1 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 β1 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 β1 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 β1 integrin or EGFR single inhibition, combined β1 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 β1 integrin inhibition. In vivo, simultaneous anti-β1 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 β1 integrin and EGFR blocking. CONCLUSIONS Concomitant targeting of β1 integrin and EGFR seems a powerful and promising approach to overcome radioresistance of HNSCCs.

The cancer cell adhesion resistome: mechanisms, targeting and translational approaches.

Abstract Cell adhesion-mediated resistance limits the success of cancer therapies and is a great obstacle to overcome in the clinic. Since the 1990s, where it became clear that adhesion of tumor cells to the extracellular matrix is an important mediator of therapy resistance, a lot of work has been conducted to understand the fundamental underlying mechanisms and two paradigms were deduced: cell adhesion-mediated radioresistance (CAM-RR) and cell adhesion-mediated drug resistance (CAM-DR). Preclinical work has evidently demonstrated that targeting of integrins, adapter proteins and associated kinases comprising the cell adhesion resistome is a promising strategy to sensitize cancer cells to both radiotherapy and chemotherapy. Moreover, the cell adhesion resistome fundamentally contributes to adaptation mechanisms induced by radiochemotherapy as well as molecular drugs to secure a balanced homeostasis of cancer cells for survival and growth. Intriguingly, this phenomenon provides a basis for synthetic lethal targeted therapies simultaneously administered to standard radiochemotherapy. In this review, we summarize current knowledge about the cell adhesion resistome and highlight targeting strategies to override CAM-RR and CAM-DR.

EGFR and β1-integrin targeting differentially affect colorectal carcinoma cell radiosensitivity and invasion.

BACKGROUND AND PURPOSE Simultaneous targeting of β1 integrin receptor and epidermal growth factor receptor (EGFR) showed higher level of radiosensitization in head and neck cancers than monotherapies. As EGFR inhibition is similarly performed in colorectal cancer (CRC), we investigated the radiosensitizing and anti-invasive potential of β1-integrin/EGFR inhibition in CRC cell lines grown in more physiological three-dimensional (3D) matrix-based cell cultures. MATERIALS AND METHODS DLD-1 and HT-29 cells were used for 3D-colony formation, invasion and proliferation assays and Western blotting. β1 integrin, focal adhesion kinase and EGFR were inhibited by AIIB2, TAE226 and Cetuximab, respectively. KRAS and BRAF knockdown were accomplished using small-interfering RNA technology. Single doses of X-rays ranged from 2Gy to 6Gy and 5-fluorouracil (5-FU) concentration was 10μM. RESULTS Neither β1-integrin/EGFR inhibition nor KRAS or BRAF depletion nor 5-FU significantly modified CRC cell radiosensitivity. Cetuximab, AIIB2 and Cetuximab/AIIB2 differentially modulated MAPK, JNK and AKT phosphorylation. AIIB2 and TAE226 significantly decreased cell invasion. CONCLUSIONS Our data show inefficiency of Cetuximab and AIIB2 on top of radiochemotherapy. The functions of KRAS and BRAF in therapy resistance remain unanswered and warrant further preclinical molecular-driven investigations. One promising approach might be β1 integrin targeting for reducing metastatic CRC cell spread.

BEMER Electromagnetic Field Therapy Reduces Cancer Cell Radioresistance by Enhanced ROS Formation and Induced DNA Damage

Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of non-invasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF) to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radiosensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease.

Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting

Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor β1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with β1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by β1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a β1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.

Whole exome sequencing identifies mTOR and KEAP1 as potential targets for radiosensitization of HNSCC cells refractory to EGFR and β1 integrin inhibition

Intrinsic and acquired resistances are major obstacles in cancer therapy. Genetic characterization is commonly used to identify predictive or prognostic biomarker signatures and potential cancer targets in samples from therapy-naïve patients. By far less common are such investigations to identify specific, predictive and/or prognostic gene signatures in patients or cancer cells refractory to a specific molecular-targeted intervention. This, however, might have a great value to foster the development of tailored, personalized cancer therapy. Based on our identification of a differential radiosensitization by single and combined β1 integrin (AIIB2) and EGFR (Cetuximab) targeting in more physiological, three-dimensional head and neck squamous cell carcinoma (HNSCC) cell cultures, we performed comparative whole exome sequencing, phosphoproteome analyses and RNAi knockdown screens in responder and non-responder cell lines. We found a higher rate of gene mutations with putative protein-changing characteristics in non-responders and different mutational profiles of responders and non-responders. These profiles allow stratification of HNSCC patients and identification of potential targets to address treatment resistance. Consecutively, pharmacological inhibition of mTOR and KEAP1 effectively diminished non-responder insusceptibility to β1 integrin and EGFR targeting for radiosensitization. Our data pinpoint the added value of genetic biomarker identification after selection for cancer subgroup responsiveness to targeted therapies.

Abstract 5838: Adhesion- and stress-related adaptation mechanisms eliciting glioblastoma radiochemoresistance can be effectively circumvented by beta1 integrin/JNK co-targeting

Glioblastoma multiforme (GBM) is the most common brain tumor in adults and characterized by poor clinical outcome due to genetic and epigenetic alterations in resistance-mediating genes and destructive infiltration into the normal brain. Upon therapy, malignant tumors show adaptation to maintain their homeostasis. Two critical determinants of this adaptation process are cell adhesion by beta1 integrins and stress signaling via c-Jun N-terminal kinases (JNK). Here, we evaluated the potential of simultaneous beta1 integrin/JNK targeting to overcome GBM adaptation controlling radiochemoresistance and invasion. Comparative Oncomine data base analysis was performed on the expression of JNK1/2/3 isoforms, beta1 integrin and its ligands in GBM with normal brain. Different human GBM cell populations (patient-derived, stem-like, established) were analyzed for sphere formation, clonogenicity, 3D collagen type-1 invasion, cell cycling, chromatin organization, DNA double strand break (DSB) repair (γH2AX foci assay), broad-spectrum phosphoproteome analysis, FACS analysis and protein expression/phosphorylation upon irradiation (0-6 Gy X-rays) and chemotherapy (Temozolomide) with and without single and simultaneous inhibition of beta1 integrin (AIIB2) and JNK (SP600125, JNKi). The radiochemosensitizing potential of AIIB2/JNKi was also investigated in an orthotopic GBM mouse model using stem-like cells. In contrast to JNK isoforms, beta1 integrin and col1 showed significant overexpression in GBM compared with normal brain. While single inhibition of beta1 integrin and JNK mediated cytotoxicity, only combined targeting resulted in radiochemosensitization. Intriguingly, double AIIB2/JNKi treatment abrogated GBM cell invasion. Importantly, dual beta1 integrin/JNK inhibition elicited a significant reduction in tumor growth and longer survival of mice concomitantly treated with radiotherapy/Temozolomide. Mechanistically, JNK blocking induced beta1 integrin expression for stimulating diverse signaling pathways controlling cell cycling, invasion and radiochemosensitivity. Radiosensitization by AIIB2/JNKi is caused by enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest as well as impaired DNA double strand break repair in the context of elevated levels of euchromatin. In summary, our data reveal that dual beta1 integrin/JNK targeting efficiently impairs adhesion and stress-related adaptation mechanisms involved in radiochemoresistance and invasion. More in-depth evaluation is warranted to clarify the potential of this kind of beta1 integrin/JNK multi-targeting strategy administrated concomitantly to standard radiochemotherapy in patients suffering from GBM. Note: This abstract was not presented at the meeting. Citation Format: Anne Vehlow, Erik Klapproth, Katja Storch, Ellen Dickreuter, Michael Seifert, Antje Dietrich, Rebecca Butof, Achim Temme, Nils Cordes. Adhesion- and stress-related adaptation mechanisms eliciting glioblastoma radiochemoresistance can be effectively circumvented by beta1 integrin/JNK co-targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5838. doi:10.1158/1538-7445.AM2017-5838

3D-Zellkultur zur Identifizierung von Zielmolekülen für die Krebstherapie

Malignant tumors often show radio- and chemoresistance phenotypes resulting from genetic mutations and epigenetic regulation. As molecular examinations for therapy optimization in patients and animal models are often challenging or infeasible, matrix-based 3-dimensional (3D) cell culture models have been developed as powerful tools in translational research to unravel the mechanisms of cancer cell survival, proliferation and metastasis as well as to identify potential cancer targets.

Abstract LB-211: beta1 integrin targeting results in radiosensitization by controlling DNA repair mechanisms and chromatin organization

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Integrin-mediated cell-extracellular matrix (ECM) interactions confer cancer cell resistance to therapy. Our own data and data of others generated in three-dimensional (3D) ECM-based cell cultures and in vivo provided evidence that particularly beta1 integrin targeting potently reduces this resistance involving downstream integrin signaling mediators like focal adhesion kinase, cortactin and JNK1. However, it remains unknown whether beta1 integrin inhibition affects the repair of radiation-induced DNA double strand breaks (DSB) and their distribution in eu- and heterochromatic DNA regions. Here, we show that 3D growth (cell culture, xenograft models) increased the amount of heterochromatin in human head and neck squamous cell (HNSCC) and lung carcinoma cell lines relative to 2D, induced higher radioresistance, reduced the number of chromosomal aberrations and gammaH2AX/53BP1-positive foci, which was accompanied by a shift of the euchromatin-to-heterochromatin DSB distribution from a 2:1 (2D) to a 1:1 (3D). Upon beta1 integrin inhibition using the inhibitory monoclonal antibody AIIB2 or siRNA, cells grown in 3D or as xenografts in nude mice were radiosensitized or showed a significant growth delay, respectively. The disruption of beta1 integrin-mediated cell-ECM interactions induced an increase in heterochromatin levels indicated by HP1alpha and H3K9me3 elevation. In line with these findings was a strong impairment of both ATM and DNA-PK and their associated downstream signaling pathways (e.g. Chk2, Mre11, Nbs1, Rad50) in beta1 integrin inhibited, irradiated cells relative to IgG isotype controls. In contrast to unchanged number of heterochromatic foci identified in stably transfected HP1alpha cells, kinetic DSB measurements revealed an increased number of euchromatic foci upon beta1 integrin blocking, which evidently showed strongly perturbed DSB repair. In conclusion, our data generated in 3D cell culture and tumor xenograft models suggest a regulatory function of beta1 integrin signaling in the repair of radiogenic DSBs and the organization of chromatin. In addition to our understanding of the initial steps deactivated upon beta1 integrin blocking, these findings bridge membrane bound integrin function to nuclear DNA repair processes, thus providing further insights into how integrins confer cancer cell radioresistance and why integrin targeting is potentially useful as adjuvant to conventional radio- and radiochemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-211. doi:1538-7445.AM2012-LB-211