The Role of the Focal Adhesion Protein PINCH1 for the Radiosensitivity of Adhesion and Suspension Cell Cultures

Focal adhesion (FA) signaling mediated by adhesion to extracellular matrix and growth factor receptors contributes to the regulation of the cellular stress response to external stimuli. Critical to focal adhesion assembly and signaling is the adapter protein PINCH1. To evaluate whether the prosurvival function of PINCH1 in radiation cell survival depends on cell adhesion, we examined PINCH1fl/fl and PINCH1−/− mouse embryonic fibroblasts and human cancer cell lines. Here, we found that the enhanced cellular radiosensitivity mediated by PINCH1 depletion observed under adhesion conditions is conserved when cells are irradiated under suspension conditions. This unsuspected finding could not be explained by the observed modification of adhesion and growth factor associated signaling involving FAK, Paxillin, p130CAS, Src, AKT, GSK3β and ERK1/2 under suspension and serum withdrawal relative to adhesion conditions with serum. Our data suggest that the adapter protein PINCH1 critically participates in the regulation of the cellular radiosensitivity of normal and malignant cells similarly under adhesion and suspension conditions

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; gammaH2AX/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

Workshop Report for Cancer Research: Defining the Shades of Gy: Utilizing the Biological Consequences of Radiotherapy in the Development of New Treatment Approaches—Meeting Viewpoint

The ability to physically target radiotherapy using image-guidance is continually improving with photons and particle therapy that include protons and heavier ions such as carbon. The unit of dose deposited is the gray (Gy); however, particle therapies produce different patterns of ionizations, and there is evidence that the biological effects of radiation depend on dose size, schedule, and type of radiation. This National Cancer Institute (NCI)–sponsored workshop addressed the potential of using radiation-induced biological perturbations in addition to physical dose, Gy, as a transformational approach to quantifying radiation

Comprehensive analysis of signal transduction in three-dimensional ECM-based tumor cell cultures

Analysis of signal transduction and protein phosphorylation is fundamental to understand physiological and pathological cell behavior as well as identification of novel therapeutic targets. Despite the fact that more physiological three-dimensional cell culture assays are increasingly used, particularly proteomics and phosphoproteomics remain challenging due to easy, robust and reproducible sample preparation. Here, we present an easy-to-perform, reliable and time-efficient method for the production of 3D cell lysates without compromising cell adhesion before cell lysis. The samples can be used for Western blotting as well as phosphoproteome array technology. This technique would be of interest for researchers working in all fields of biology and drug development

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

Plating efficiencies of <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− MEFs under indicated growth conditions.

<p>*Poly-S, polystyrene; Poly-L, poly-L-lysine; FN, Fibronectin.</p

Signal transduction modification in adherent and suspension tumor cell lines after PINCH1 knockdown.

<p>(A) Western blot on total cell lysates from PINCH1 depleted HTB43 and HTB35 cells grown under adhesion or in suspension. (B) Densitometric analysis from protein bands shown in ‘A’ after normalization to total protein or β-Actin expression and subsequently to adhesion conditions of siRNA control cells ( = 1). Co, control; P1, PINCH1.</p

Apoptosis in irradiated cells remains unchanged by PINCH1 silencing.

<p>(A) and (B) HTB43 and HTB35 were assessed for typical apoptotic nuclear morphology upon X-ray irradiation (0 or 6 Gy) and transfection with PINCH1 specific or non-specific control siRNA (mean±SD; <i>n</i> = 3).</p

Effects of PINCH1 knockout on signal transduction under adhesion and suspension conditions.

<p>(A) Western blot on total cell lysates from <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− MEF grown on polystyrene plus FCS (first lane), in suspension plus FCS (second lane) or in suspension without FCS (third lane). (B) Densitometric analysis from protein bands shown in ‘A’ after normalization to total protein or β-Actin expression and subsequently to adhesion plus FCS conditions of <i>PINCH1</i>^fl/fl MEF ( = 1). FCS, fetal calf serum; Susp, suspension.</p

Colony cell numbers and cell morphology remain unaltered upon PINCH1 depletion.

<p>(A) and (B) Cell numbers of 15 colonies were counted in PINCH1 knockdown and control HTB43 and HTB35 cell cultures. Results show mean±SD (<i>n</i> = 3; t-test; n.s., not significant). (C) and (D) show cellular morphology in PINCH1 depleted and control cell colonies of HTB43 and HTB35 tumor cell lines. Photographs illustrate (<i>i</i>) representative colony growth in 35-mm wells, (<i>ii</i>) a single representative colony (magnification = 10×), and (<i>iii</i>) a representative zoom (magnification = 40×) from the edge of a colony. Co, control.</p