GLI1, CTNNB1 and NOTCH1 protein expression in a thymic epithelial malignancy tissue microarray.

BACKGROUND/AIM: Thymic epithelial tumors (TET) are rare. Wingless and INT (WNT), NOTCH and sonic hedgehog pathway interactions between thymocytes and thymic stroma are important to thymus and T-cell development. We analyzed a thymoma tissue microarray (TMA) for glioma associated oncogene homolog 1 (GLI1), NOTCH1 and catenin (cadherin-associated protein, beta 1) (CTNNB1) expression as surrogate markers of sonic hedgehog, NOTCH and WNT pathway activity. MATERIALS AND METHODS: GLI1, NOTCH1 and CTNNB1 expression were assayed in a tissue microarray of 68 TET and eight benign thymus by fluorescent immunohistochemistry (AQUA) as surrogates for activity of the sonic hedgehog, NOTCH and WNT pathways respectively. RESULTS: No difference in tumor GLI1 (mean 201 vs. 211, p=0.31), CTNNB1 (mean 222 vs. 306, p=0.66) or NOTCH1 expression (mean 317 vs. 325, p=0.82) was noted between thymic tumor and benign thymus. CONCLUSION: No evidence for preferential expression of GLI1, NOTCH1 or CTNNB1 was noted. High-throughput immunofluorescence using AQUA technology can help overcome limitations of small sample size and tissue heterogeneity when analyzing protein expression in thymic tumors.

Interaction of Hsp90 with the nascent form of the mutant epidermal growth factor receptor EGFRvIII.

EGFRvIII is a mutant epidermal growth factor that promotes aggressive growth of glioblastomas. We made a plasmid that directed the expression of an EGFRvIII with three copies of the Flag epitope at its amino terminus. Flag-tagged EGFRvIII was expressed at the same levels as unmodified EGFRvIII, and showed the same subcellular localization. However, the Flag epitope could only be detected on EGFRvIII present in the endoplasmic reticulum; the epitope was covalently modified during trafficking of the receptor through the Golgi so that it was no longer recognized by anti-Flag antibody. This property was exploited to selectively purify nascent EGFRvIII from glioblastoma cells. Nascent EGFRvIII was found to copurify with a set of other proteins, identified by mass spectrometry as the two endoplasmic reticulum chaperones Grp94 and BiP, and the two cytosolic chaperones Hsc70 and Hsp90. The Hsp90-associated chaperone Cdc37 also co-purified with EGFRvIII, suggesting that Hsp90 binds EGFRvIII as a complex with this protein. Geldanamycin and radicicol, two chemically unrelated inhibitors of Hsp90, decreased the expression of EGFRvIII in glioblastoma cells. These studies show that nascent EGFRvIII in the endoplasmic reticulum associates with Hsp90 and Cdc37, and that the Hsp90 association is necessary to maintain expression of EGFRvIII.