IGF-1R inhibition : A tool for functional studies of insulin-like growth factor family in malignant cells

Sammanfattning: Cancer cells generally posses the capability of overusing normal extracellular signaling for proliferation and/or antiapoptosis to create growth advantage over the normal cells. Major players in extracellular signaling are the growth factor receptors. Among them, an activated IGF-1R is important for the establishment of a malignant cell phenotype. Interestingly, the targeting of IGF-1R can reverse the malignant phenotype in cancer cells and render them sensitive to apoptosis, without seriously affecting the biology of normal cells. For these reasons, IGF-1R seems to be a very promising target in cancer therapy. Recently, we demonstrated that the cyclolignan PPP efficiently inhibited phosphorylation of IGF-1R without interfering with insulin receptor activity. This thesis is centered on: (1) functional studies of IGF-1R using PPP as a tool, with focus on importance for survival and proliferation of malignant cells as well as possible mechanisms of PPP action; (2) possible caveats in clinical applications of PPP (e.g. resistance, side effects secondary to IGF-1R inhibition, effects on glucose uptake). Using a IGF-1R tyrosine kinase construct, isolated by immunoprecipitation and amplified in insect cells, we found that PPP decreased phosphorylation of tyrosine residue (Y) 1136 in the activation loop of the IGF-1R kinase domain. Studies using dominant-negative constructs of IGF-1R (in which specific tyrosine residues are replaced by phenylalanine) suggest that the inhibition of Y 1136 phosphorylation may be important for the inhibition of Akt phosphorylation seen in PPP treated cell cultures. Whether PPP directly or indirectly (e.g. by interfering with IGF-1R associated proteins) inhibits Y1136 phosphorylation is still unknown. It was confirmed that inhibitions of downstream reactions of the phosphatyl inositol-3 kinase/anti-apoptotic pathway (e.g. attenuated Bad phosphorylation, PARP cleavage, caspase activation) were a consequence of the PPP-induced inhibition of IGF-1R. (Paper 1) We demonstrated the presence and growth dependence of IGF-1R in primary cultured craniopharyngioma cells from a subset of affected patients (5 out of 9). Upon treatment with PPP, cells with high IGF-1R expression responded promptly with decreased Akt phosphorylation followed by cell growth inhibition, whereas these responses did not appear in cells with low receptor expression. Our data points to the possibility of using IGF-1R inhibitors (e.g. PPP) as a treatment modality to obtain complete tumor-free conditions before growth hormone substitution. (Paper 2) A general concern with antitumor agents is development of resistance. In light of this problem we aimed to investigate whether malignant cells may develop serious resistance to PPP. After trying to select several malignant cell lines, only two out of 10 survived an 80-week selection. We could observe a temporary and limited increase in IGF- I R expression but there were no rearrangements or amplification of the IGF-1R gene. The resistant cell lines did not exhibit cross-resistance to known cytostatic dugs. In conclusion, no or slight resistance to PPP occurred. (Paper 3) Finally, we confirmed that PPP does not inhibit activity of the highly related insulin receptor and induce diabetogenic effects (like high blood glucose). Instead, in vivo and in vitro studies showed that PPP treatment reduces the blood glucose levels in mice and induces increase of glucose uptake in cells expressing the insulin-dependent glucosetransporter GLUT-4. (Paper 4)