title: 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-mTOR(Ser2481) 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.
authors: Department of Pathology, Dr Josep Trueta University Hospital of Girona, Catalonia, Spain.
authors: Eugeni
authors: E
authors: Lopez-Bonet
authors: Alejandro
authors: A
authors: Vazquez-Martin
authors: Maria Carmen
authors: MC
authors: Pérez-Martínez
authors: Cristina
authors: C
authors: Oliveras-Ferraros
authors: Ferran
authors: F
authors: Pérez-Bueno
authors: Luis
authors: L
authors: Bernadó
authors: Javier A
authors: JA
authors: Menendez
mesh_terms: D001706:Biopsy; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology*; D002467:Cell Nucleus / Q000378:metabolism; D002875:Chromosomes / Q000648:ultrastructure*; D003593:Cytoplasm / Q000378:metabolism; D005819:Genetic Markers; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods*; D008938:Mitosis; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000737:chemistry*; D058570:TOR Serine-Threonine Kinases
chemical_list: D005819:Genetic Markers; D047908:Intracellular Signaling Peptides and Proteins; D012694:Serine; C546842:MTOR protein, human; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases