Serine 2481-autophosphorylation of mammalian target of rapamycin (mTOR) couples with chromosome condensation and segregation during mitosis: Confocal microscopy characterization and immunohistochemical validation of PP-mTORSer2481 as a novel high-contrast mitosis marker in breast cancer core biopsies

Abstract

Abstract. The prognostic abilities of breast cancer gene expression signatures are due mostly to the detection of proliferation activity. One of the strongest, yet simple and well-reproducible proliferation-associated prognostic factors is the mitotic activity index (MAI). However: a) counting mitotic figures is regarded by many histopathologists as cumbersome and time-consuming, and b) most available immunohistochemical markers are much weaker predictors than the MAI. We have investigated the spatio-temporal sub-cellular distribution of the Serine 2481-autophosphorylated form of mTOR (PP-mTOR Ser2481 ) during the G 1 /S-to-M-phase transition both in cultured cancer cells and in cancer tissue specimens. Using a high-resolution, automated confocal high-content imaging system, we observed that mitotic cells notably accumulated a distinct pattern of nuclear and cytoplasmic immunolabelings of PP-mTOR Ser2481 . Parallel experiments examining site-specific phosphorylation (i.e., Serine 10 and Serine 28) of the G 2 /M marker Histone H3 (PP-H3) revealed that PP-H3 Ser10/Ser28 staining efficiently detected mitotic cells from prophase until the beginning of anaphase, but not during late anaphase, telophase and cytokinesis. PP-mTOR Ser2481 staining associated near and between separating chromosomes not only during early mitotic stages but also to the midzone and to midbody at ana/telophase through cytokinesis. We then evaluated the usefulness of PP-mTOR Ser2481 immunostaining for improving the efficiency of mitotic counting using. Anti-PPmTOR Ser2481 -labeled mitotic figures (MFs) were easily seen and permitted a quick identification of mitotic hotspots in formalinfixed cancer tissues, even at low magnification. Importantly, average mitotic counts were significantly higher when using PP-mTOR Ser2481 staining than with the hematoxylin and eosin (H&E) protocol in breast cancer core biopsies. Mitotic count based on PP-mTOR Ser2481 immunostaining increased tumor grade by one grade in 2 of 9 breast carcinomas. These findings warrant forthcoming studies to confirm both the accuracy and the prognostic value of PP-mTOR Ser2481 as a novel high-contrast immunohistochemical mitosis marker in larger populations of human breast carcinomas.