Iris Eke

β1 Integrin/FAK/cortactin signaling is essential for human head and neck cancer resistance to radiotherapy

Integrin signaling critically contributes to the progression, growth, and therapy resistance of malignant tumors. Here, we show that targeting of β₁ integrins with inhibitory antibodies enhances the sensitivity to ionizing radiation and delays the growth of human head and neck squamous cell carcinoma cell lines in 3D cell culture and in xenografted mice. Mechanistically, dephosphorylation of focal adhesion kinase (FAK) upon inhibition of β₁ integrin resulted in dissociation of a FAK/cortactin protein complex. This, in turn, downregulated JNK signaling and induced cell rounding, leading to radiosensitization. Thus, these findings suggest that robust and selective pharmacological targeting of β₁ integrins may provide therapeutic benefit to overcome tumor cell resistance to radiotherapy.

Three-Dimensional Cell Growth Confers Radioresistance by Chromatin Density Modification

Cell shape and architecture are determined by cell-extracellular matrix interactions and have profound effects on cellular behavior, chromatin condensation, and tumor cell resistance to radiotherapy and chemotherapy. To evaluate the role of chromatin condensation for radiation cell survival, tumor cells grown in three-dimensional (3D) cell cultures as xenografts and monolayer cell cultures were compared. Here, we show that increased levels of heterochromatin in 3D cell cultures characterized by histone H3 deacetylation and induced heterochromatin protein 1alpha expression result in increased radiation survival and reduced numbers of DNA double strand breaks (DSB) and lethal chromosome aberrations. Intriguingly, euchromatin to heterochromatin-associated DSBs were equally distributed in irradiated 3D cell cultures and xenograft tumors, whereas irradiated monolayer cultures showed a 2:1 euchromatin to heterochromatin DSB distribution. Depletion of histone deacetylase (HDAC) 1/2/4 or application of the class I/II pharmacologic HDAC inhibitor LBH589 induced moderate or strong chromatin decondensation, respectively, which was translated into cell line-dependent radiosensitization and, in case of LBH589, into an increased number of DSBs. Neither growth conditions nor HDAC modifications significantly affected the radiation-induced phosphorylation of the important DNA repair protein ataxia telangiectasia mutated. Our data show an interrelation between cell morphology and cellular radiosensitivity essentially based on chromatin organization. Understanding the molecular mechanisms by which chromatin structure influences the processing of radiation-induced DNA lesions is of high relevance for normal tissue protection and optimization of cancer therapy.

Radiobiology goes 3D: How ECM and cell morphology impact on cell survival after irradiation

Translational research is essential to find new therapeutic approaches to improve cancer patient survival. Despite extensive efforts in preclinical studies, many novel therapies fail to turn out to be translational from bench to beside. Therefore, new models better reflecting the conditions in vivo are needed to generate results, which transfer reliably into the clinic. The use of three-dimensional (3D) cell culture models has provided new emerging insights into the understanding of cellular behavior upon cancer therapies. Interestingly, cells cultured in a 3D extracellular matrix are more radio- and chemoresistant than cells grown under conventional 2D conditions. In this review, we summarize and discuss underlying mechanisms of this phenomenon including integrin-mediated cell-matrix interactions, cell shape, nuclear organization and chromatin structure. Identifying the molecular differences between 2D and 3D cultured cells will offer the opportunity to improve our research and widen our therapeutic possibilities against cancer.

PINCH1 regulates Akt1 activation and enhances radioresistance by inhibiting PP1α

Tumor cell resistance to ionizing radiation and chemotherapy is a major obstacle in cancer therapy. One factor contributing to this is integrin-mediated adhesion to ECM. The adapter protein particularly interesting new cysteine-histidine-rich 1 (PINCH1) is recruited to integrin adhesion sites and promotes cell survival, but the mechanisms underlying this effect are not well understood. Here we have shown that PINCH1 is expressed at elevated levels in human tumors of diverse origins relative to normal tissue. Furthermore, PINCH1 promoted cell survival upon treatment with ionizing radiation in vitro and in vivo by perpetuating Akt1 phosphorylation and activity. Mechanistically, PINCH1 was found to directly bind to protein phosphatase 1alpha (PP1alpha) - an Akt1-regulating protein - and inhibit PP1alpha activity, resulting in increased Akt1 phosphorylation and enhanced radioresistance. Thus, our data suggest that targeting signaling molecules such as PINCH1 that function downstream of focal adhesions (the complexes that mediate tumor cell adhesion to ECM) may overcome radio- and chemoresistance, providing new therapeutic approaches for cancer.

Caveolin-1 mediated radioresistance of 3D grown pancreatic cancer cells.

BACKGROUND AND PURPOSE Resistance of pancreatic ductal adenocarcinoma (PDAC) to chemo- and radiotherapy is a major obstacle. The integral membrane protein Caveolin-1 (Cav-1) has been suggested as a potent target in human pancreatic carcinoma cells. MATERIALS AND METHODS Human pancreatic tumor cells were examined in a three-dimensional (3D) cell culture model with regard to clonogenic survival, apoptosis, radiogenic DNA-double strand breaks and protein expression and phosphorylation under siRNA-mediated knockdown of Cav-1 without and in combination with irradiation (X-rays, 0-6Gy). Immunohistochemistry was used to assess Cav-1 expression in biopsies from patients with PDAC. RESULTS Tumor cells in PDAC showed significantly higher Cav-1 expression relative to tumor stroma. Cav-1 knockdown significantly reduced beta1 integrin expression and Akt phosphorylation, induced Caspase 3- and Caspase 8-dependent apoptosis and enhanced the radiosensitivity of 3D cell cultures. While cell cycling and Cav-1 promoter activity remained stable, Cav-1 knockdown-induced radiosensitization correlated with elevated numbers of residual DNA-double strand breaks. CONCLUSIONS Our data strongly support the concept of Cav-1 as a potent target in pancreatic carcinoma cells due to radiosensitization and Cav-1 overexpression in tumor cells of PDAC. 3D cell cultures are powerful and useful tools for the testing of novel targeting strategies to optimize conventional radio- and chemotherapy regimes for PDAC.

Focal adhesion signaling and therapy resistance in cancer

Interlocking gene mutations, epigenetic alterations and microenvironmental features perpetuate tumor development, growth, infiltration and spread. Consequently, intrinsic and acquired therapy resistance arises and presents one of the major goals to solve in oncologic research today. Among the myriad of microenvironmental factors impacting on cancer cell resistance, cell adhesion to the extracellular matrix (ECM) has recently been identified as key determinant. Despite the differentiation between cell adhesion-mediated drug resistance (CAMDR) and cell adhesion-mediated radioresistance (CAMRR), the underlying mechanisms share great overlap in integrin and focal adhesion hub signaling and differ further downstream in the complexity of signaling networks between tumor entities. Intriguingly, cell adhesion to ECM is per se also essential for cancer cells similar to their normal counterparts. However, based on the overexpression of focal adhesion hub signaling receptors and proteins and a distinct addiction to particular integrin receptors, targeting of focal adhesion proteins has been shown to potently sensitize cancer cells to different treatment regimes including radiotherapy, chemotherapy and novel molecular therapeutics. In this review, we will give insight into the role of integrins in carcinogenesis, tumor progression and metastasis. Additionally, literature and data about the function of focal adhesion molecules including integrins, integrin-associated proteins and growth factor receptors in tumor cell resistance to radio- and chemotherapy will be elucidated and discussed.

The Small Molecule Inhibitor QLT0267 Radiosensitizes Squamous Cell Carcinoma Cells of the Head and Neck

Background The constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC). Methodology/Principal Findings Parental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK floxed/floxed(fl/fl) and ILK −/− mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0–6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; γH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK fl/fl fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls. Conclusions/Significance Our data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic.

3D cell cultures of human head and neck squamous cell carcinoma cells are radiosensitized by the focal adhesion kinase inhibitor TAE226

BACKGROUND AND PURPOSE Focal adhesion kinase (FAK), a main player in integrin signaling and survival, is frequently overexpressed in human cancers and therefore postulated as potential target in cancer therapy. The aim of this study was to evaluate the radiosensitizing potential of the FAK inhibitor TAE226 in three-dimensional (3D) tumor cell cultures. MATERIALS AND METHODS Head and neck squamous cell carcinoma (HNSCC) cells (FaDu, UT-SCC15, UT-SCC45), lung cancer cells (A549), colorectal carcinoma cells (DLD-1, HCT-116) and pancreatic tumor cells (MiaPaCa2, Panc1) were treated with different concentrations of TAE226 (0-1mum; 1 or 24h) without or in combination with irradiation (0-6Gy, X-ray, single dose). Subsequently, 3D clonogenic survival assays (laminin-rich extracellular matrix) and Western blotting (expression/phosphorylation, e.g. FAK, Akt, ERK1/2) were performed. RESULTS All investigated 3D cell cultures showed a dose-dependent reduction in clonogenic survival by TAE226. Intriguingly, TAE226 only significantly radiosensitized 3D HNSCC cell cultures accompanied by a pronounced dephosphorylation of FAK, Akt and ERK1/2. CONCLUSIONS Our data demonstrate TAE226 as potent FAK inhibitor that enhances the cellular radiosensitivity particularly of HNSCC cells grown in a 3D cell culture model. Future in vitro and in vivo investigations will clarify, to which extent this approach might be clinically relevant for radiotherapy of HNSCC.

EGFR/JIP-4/JNK2 Signaling Attenuates Cetuximab-Mediated Radiosensitization of Squamous Cell Carcinoma Cells

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-NH(2)-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.

Pharmacological inhibition of EGFR tyrosine kinase affects ILK-mediated cellular radiosensitization in vitro

Purpose: Integrin-linked kinase (ILK) mediates signals from β integrins and links integrins to epidermal growth factor receptor (EGFR). Previous studies have identified an antisurvival effect of ILK in irradiated cells. The aim of this study was to evaluate the role of EGFR tyrosine kinase (tk) activity for ILK-mediated radiosensitization. Materials and methods: Human FaDu squamous cell carcinoma (SCC) cells stably transfected with hyperactive ILK (ILK-hk) and ILKfl/fl and ILK−/− mouse fibroblasts were treated with the pharmacological EGFR-tk inhibitor BIBX1382BS without or in combination with single doses of X-rays. Clonogenic radiation survival, protein expression and phosphorylation (EGFR, v-akt murine thymoma viral oncogene homolog 1 (Akt), p42/44 mitogen-activated protein kinase), DNA-double strand break (DSB) repair measured by γH2AX foci, cell morphology and cell cycle distribution were examined. Results: Expression of ILK-hk or ILKfl/fl status resulted in significant radiosensitization relative to vector controls or ILK−/−. Following BIBX1382BS, clonogenic survival of normal fibroblasts and vector controls remained unaffected while ILK-hk-related radiosensitization was significantly diminished. In contrast to BIBX1382BS, which did not affect DNA-DSB repair, ILK-hk-mediated radiosensitization was associated with reduced DNA-DSB repair. At 10 days after BIBX1382BS treatment, FaDu transfectants, in contrast to fibroblasts, showed reduced cell size, accumulation of G1 phase cells and reduced Akt-serine(S)473 phosphorylation. Conclusions: Our findings confirm ILK as a cell type-independent antisurvival factor in irradiated cells, which actions in terms of radiosensitization critically depend on proper EGFR-tk activity.