The role of TGF-β in the development of thyrocyte hyperplasia in NOD.H2h4 mice [abstract]

Faculty Mentor: Helen Mullen, ImmunologyAbstract only availableWild type (WT) NOD.H-2h4 mice develop lymphocytic spontaneous autoimmune thyroiditis (L-SAT) and IFN-γ-/- NOD.H-2h4 mice develop severe thyroid epithelial cell (TEC) hyperplasia when given 0.05% NaI water. Since hyperplastic TEC in IFN-γ-/- mice strongly express TGF-β, transgenic NOD.H-2h4 mice expressing TGF-β on TEC were generated to test the hypothesis that overexpression of TGF-β on TEC would promote earlier and/or more severe TEC hyperplasia. Consistent with this hypothesis, all IFN-γ-/- NOD.H-2h4 mice developed severe thyrocyte hyperplasia, and compared to WT Tg- mice with L-SAT, all WT Tg+ mice developed thyrocyte hyperplasia with minimal lymphocyte infiltration 2 months after NaI water. The goal of this study was to compare lymphocyte activation in WT transgenic and nontransgenic mice to determine the mechanisms by which overexpression of TGF-β in thyroids inhibits L-SAT in TGF-β transgenic WT mice. Flow cytometry indicated that CD4 and CD8 splenic T-cells from WT Tg- mice with L-SAT and WT Tg+ mice with severe hyperplasia were similarly activated. By RT-PCR, splenocytes of WT Tg+ mice expressed slightly higher levels of TGF-β compared to WT Tg- mice. However, other cytokines did not differ significantly between WT Tg+ and WT Tg- mice, suggesting lymphocytes in both groups were activated to a similar extent. Splenocytes from both WT Tg+ and WT Tg- mice induced L-SAT after transfer to NOD.H-2h4 SCID recipients, suggesting splenocytes from Tg+ mice were activated and could induce L-SAT in Tg- recipients. RT-PCR and immunohistochemical staining showed that thyroids of WT Tg+ mice expressed more TGF-β and less IFN-γ than WT Tg- thyroids. These results suggest that overexpression of TGF-β on thyrocytes inhibits L-SAT and promotes thyrocyte hyperplasia in NOD.H-2h4 mice. Further research is needed to determine the mechanism by which TGF-β mediates these effects

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

Background: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. Methods: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. Results: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. Conclusions: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

Background: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells.Methods: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies.Results: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model.Conclusions: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.Peer reviewedBiochemistry and Molecular Biolog