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

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-PP-mTOR(Ser2481)-labeled mitotic figures (MFs) were easily seen and permitted a quick identification of mitotic hotspots in formalin-fixed 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.