Stephanie Hehlgans

Genome-Wide Gene Expression Analysis in Cancer Cells Reveals 3D Growth to Affect ECM and Processes Associated with Cell Adhesion but Not DNA Repair

Cell morphology determines cell behavior, signal transduction, protein-protein interaction, and responsiveness to external stimuli. In cancer, these functions profoundly contribute to resistance mechanisms to radio- and chemotherapy. With regard to this aspect, this study compared the genome wide gene expression in exponentially growing cell lines from different tumor entities, lung carcinoma and squamous cell carcinoma, under more physiological three-dimensional (3D) versus monolayer cell culture conditions. Whole genome cDNA microarray analysis was accomplished using the Affymetrix HG U133 Plus 2.0 gene chip. Significance analysis of microarray (SAM) and t-test analysis revealed significant changes in gene expression profiles of 3D relative to 2D cell culture conditions. These changes affected the extracellular matrix and were mainly associated with biological processes like tissue development, cell adhesion, immune system and defense response in contrast to terms related to DNA repair, which lacked significant alterations. Selected genes were verified by semi-quantitative RT-PCR and Western blotting. Additionally, we show that 3D growth mediates a significant increase in tumor cell radio- and chemoresistance relative to 2D. Our findings show significant gene expression differences between 3D and 2D cell culture systems and indicate that cellular responsiveness to external stress such as ionizing radiation and chemotherapeutics is essentially influenced by differential expression of genes involved in the regulation of integrin signaling, cell shape and cell-cell contact.

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.

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.

Expression of Integrin-linked Kinase Is Increased in Differentiated Cells

Integrin-linked kinase (ILK), a mediator of β integrin signals, has emerged as a therapeutic target in malignant tumors. Because malignant transformation is accompanied by dedifferentiation, ILK expression was evaluated in diverse normal and tumor tissue samples with regard to tissue differentiation. In single sections and in a tissue microarray (323 tumor tissues, 181 normal tissues), immunohistochemistry was performed [ILK, Akt, phospho-Akt-S473, loricrin, transforming growth factor β2 (TGFβ2)], and staining intensities were semiquantitatively scored. Increased ILK expression was clearly associated with increased differentiation in normal gastrointestinal, neural, bone marrow, renal tissue, and in more differentiated areas of malignant tumors. ILK colocalized with its putative downstream target Akt and with loricrin or TGFβ2. Our findings clearly show that elevated levels of ILK are associated with cellular differentiation in high turnover tissues but not generally with a malignant phenotype. Our study indicates that ILK is not a general molecular target for cancer therapy but rather an indicator of differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

3D matrix-based cell cultures: Automated analysis of tumor cell survival and proliferation

Three-dimensional ex vivo cell cultures mimic physiological in vivo growth conditions thereby significantly contributing to our understanding of tumor cell growth and survival, therapy resistance and identification of novel potent cancer targets. In the present study, we describe advanced three-dimensional cell culture methodology for investigating cellular survival and proliferation in human carcinoma cells after cancer therapy including molecular therapeutics. Single cells are embedded into laminin-rich extracellular matrix and can be treated with cytotoxic drugs, ionizing or UV radiation or any other substance of interest when consolidated and approximating in vivo morphology. Subsequently, cells are allowed to grow for automated determination of clonogenic survival (colony number) or proliferation (colony size). The entire protocol of 3D cell plating takes ~1 h working time and pursues for ~7 days before evaluation. This newly developed method broadens the spectrum of exploration of malignant tumors and other diseases and enables the obtainment of more reliable data on cancer treatment efficacy.

An essential role of integrin-linked kinase in the cellular radiosensitivity of normal fibroblasts during the process of cell adhesion and spreading

Purpose: In addition to focal adhesion kinase (FAK), Paxillin and p130 Crk-associated substrate (p130Cas), integrin-linked kinase (ILK) mediates signals from β integrins for controlling, e.g., survival, adhesion and spreading. To evaluate the role of ILK in the cellular radiosensitivity at different stages of cell adhesion and spreading, ILKfloxed/floxed (fl/fl) and ILK−/− mouse fibroblasts were examined. Materials and methods: Cells were irradiated (0 – 4 Gy, X-rays) in suspension, after varying time periods on fibronectin (FN) or after 24 h on different matrix proteins. Irradiation was combined with phosphatidylinositol-3 kinase (PI3K) inhibition using Ly294002. Clonogenic radiation survival, cell adhesion, and kinetics of protein expression and phosphorylation during FN adhesion (ILK, v-akt murine thymoma viral oncogene homolog 1 (AKT), FAK, Paxillin, p130Cas) were examined. Results: In suspension and during the first hour on FN, irradiated ILKfl/fl cells survived significantly better than ILK−/− cells in a PI3K- and serum-dependent manner. 24-h cell cultures on different matrix proteins showed no difference in radiosensitivity. During FN adhesion, which was slightly impaired in ILK−/− cells, protein kinetics uncovered differences in AKT, FAK, Paxillin and p130Cas phosphorylation in the two cell lines. Phosphorylation of FAK, Paxillin and p130Cas was downregulated upon exposure to ionizing radiation in an ILK-independent manner. Conclusions: These findings indicate a critical function of ILK in the cellular radiosensitivity during the early stages of adhesion to and spreading on FN. On the basis of the presented data, a precise correlation of adhesion-, serum- and PI3K-mediated changes in PI3K/AKT and FAK/Paxillin/p130Cas signaling cascades was not found. However, identifying the underlying mechanisms of adhesion- and spreading-related changes in the cellular radiosensitivity might be relevant for an optimization of radiotherapeutic strategies specifically targeting cells located at the invasive edge of a malignant tumor.

There's something about ILK.

Purpose: Integrin-Linked Kinase (ILK) is associated with integrin and growth factor receptor signalling. As both signalling pathways contribute to cancer cell resistance, ILK seems well suited as a promising tumour target. Material and methods: Data were obtained by performing a PubMed database search and summarised with a focus on the function of ILK in cancer biology. Results: The findings on the catalytic function of ILK, on the putative substrates of ILK and on the expression of ILK in tumour and normal tissues are heterogeneous. In the context of cancer, two of these issues might be of importance. First, a variety of reports indicate a lack of ILK overexpression in tumours. Second, wild-type or overexpression of ILK has been found to considerably sensitise tumour cells to ionising irradiation as compared to ILK knockout or ILK knockdown conditions. In contrast, wild-type or overexpression of ILK has been shown to protect tumour cells from chemotherapy-induced cell death. Conclusions: Due to these conflicting data, it is difficult to evaluate if therapeutic targeting of ILK is a reasonable strategy in cancer therapy. A more comprehensive understanding of the molecular mechanisms controlled by ILK may help to answer this question.

Human head and neck squamous cell carcinoma cell lines are differentially radiosensitised by the honeybee product Propolis

Purpose: Propolis, a product of honeybees, has anti-tumoural, cytotoxic, anti-metastatic and anti-inflammatory properties. The aim of this study was the evaluation of the radiosensitising capacity of Propolis in human head and neck squamous cell carcinoma (HNSCC) cells. Materials and methods: HNSCC cell lines (FaDu, UT-SCC15, UT-SCC45), fibroblasts (HSF2) and keratinocytes (HaCaT) were treated with Propolis (0–250 μg/ml; 1, 4, 24 h) without and in combination with X-rays (0–6 Gy, single dose). Clonogenic survival, proliferation, apoptosis, expression and phosphorylation of different signalling proteins were determined. Liquid chromatography-mass spectrometry (LC-MS) was performed on Propolis. Results: Propolis significantly (P < 0.01) reduced cell growth and clonogenic survival in a time- and concentration-dependent manner. Propolis-induced apoptosis and Caspase 3 cleavage, increased phosphorylation of Extracellular signal Regulated Kinase 1/2 (ERK1/2), protein kinase B/Akt1 (Akt1) and Focal adhesion kinase (FAK). While a 1-h Propolis pretreatment was ineffective, a 3-h pretreatment significantly (P < 0.05) radiosensitised FaDu cells. LC-MS analysis identified 14 compounds of Propolis. Conclusions: Our data show that Propolis exerts cytotoxicity in a concentration- and time-dependent manner. In one out of three HNSCC cell lines, Propolis also caused an enhancement of radiosensitivity. Future studies on Propolis will shed further light on its potential as an adjuvant to radiotherapy.

Adhesion, Invasion, Integrins, and Beyond

The importance of the tumor microenvironment for tumor development and progression becomes increasingly evident. A precise and concerted interplay between both the cellular components, i.e., the cells and their mutations, and the acellular components, i.e., the tumor microenvironment, drives tumor growth and spread beyond physiological boundaries as well as promotes cellular resistance to conventional radiotherapy and chemotherapy. One of the prominent microenvironmental modulators of the sensitivity of tumor tissue and tumor-associated normal tissue to therapy is the interaction of cells with the extracellular matrix. Besides serving as structural support for the cells in a tissue, the extracellular matrix participates in the regulation of essential cell functions such as survival, proliferation, differentiation, adhesion, and migration. In this chapter, the overarching function of the tumor-related extracellular matrix is depicted and summarized with regard to the molecular, pathophysiological, and radiobiological aspects associated with tumor biology, radiation, and chemoresistance in the context of cell adhesion molecule families, their interactions with other types of cell surface receptors, and the downstream network of signal transducers.