Integrin-linked kinase: dispensable for radiation survival of three-dimensionally cultured fibroblasts.
ABSTRACT Cancer treatment by conventional radiotherapy is limited by normal tissue side-effects. Fibroblasts as "non-target" stromal cell type are considered as strong promoter of tumor growth and for developing a therapy resistant phenotype. Regarding application of novel molecular therapeutics combined with radiotherapy, evaluation of a specific targeted molecule in both tumor and normal cells is mandatory for efficacy and tolerability assessment. Previous work showed integrin-linked kinase (ILK), a mediator of beta-integrin signals and putative phosphorylator of AKT, as potent anti-survival regulator in human cancer cell lines.
To evaluate the role of ILK in normal fibroblast survival, ILK-wild-type (ILK(fl/fl)), ILK(-/-) and ILK(N-terminal) and ILK(C-terminal) domain expressing fibroblasts were irradiated with X-rays on different substrata or in three-dimensional laminin-rich extracellular matrix (lrECM).
On control substrata, ILK-deficient and ILK-mutant fibroblasts showed significant increase in radiation survival relative to ILK-wild-type cells. This effect was compensated by growth on ECM proteins and in 3D lrECM. ILK regulated AKT activity in a phosphatidylinositol-3 kinase (PI3K)-dependent manner. Upon PI3K inhibition, only ILK-wild-type fibroblasts showed significant radiosensitization.
These findings obtained in 3D cell cultures suggest ILK to be dispensable for the radiation survival response of normal fibroblasts. However, targeting the PI3K/AKT signaling axis pharmacologically might be critical for survival of normal fibroblasts exposed to ionizing radiation.
Article: The small molecule inhibitor QLT0267 Radiosensitizes squamous cell carcinoma cells of the head and neck.[show abstract] [hide abstract]
ABSTRACT: 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). 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. 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.PLoS ONE 02/2009; 4(7):e6434. · 4.09 Impact Factor