Increased understanding of the molecular heterogeneity that is intrinsic to the various subtypes of breast cancer will likely shape the future of breast cancer diagnosis, prognosis, and treatment. Advances in the field over the last several decades have been remarkable and have clearly translated into better patient care as evidenced by the earlier detection, better prognosis, and new targeted therapies. There have been two recent advances in the breast cancer research field that have lead to paradigm shifts: first, the identification of intrinsic breast tumor subtypes, which has changed the way we think about breast cancer and second, the recent characterization of cancer stem cells (CSCs), which are suspected to be responsible for tumor initiation, recurrence and resistance to therapy, have opened new exciting avenues to think about breast cancer therapeutic strategies. While these advances constitute major paradigm shifts within the research realm, the clinical arena has yet to adopt and apply our understanding of the molecular basis of the disease to early diagnosis, prognosis and therapy of breast cancers. Here, we will review the current clinical approach to classification of breast cancers, newer molecular-based classification schemes, and potential future of biomarkers representing a functional classification of breast cancer.

FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action.

Emerging evidence supports the idea that a signaling pathway containing orthologs of at least mammalian NudE and Nudel, Lis1, and cytoplasmic dynein is conserved for eukaryotic nuclear migration. In mammals, this pathway has profound impact on neuronal migration during development of the central nervous system. Lis1 and dynein are also involved in other cellular functions, such as mitosis. Here we show that Nudel also participates in a subset of dynein function in M phase. Nudel was specifically phosphorylated in M phase in its serine/threonine phosphorylation motifs, probably by Cdc2 and also Erk1 and -2. A fraction of Nudel bound to centrosomes strongly in interphase and localized to mitotic spindles in early M phase. By using mutants incapable of or simulating phosphorylation, we confirmed that phosphorylation of Nudel regulated the cellcycle-dependent distribution, possibly by increasing its dissociation rate at the microtubule-organizing center. Moreover, phosphorylated Nudel or the phosphorylation-mimicking mutant bound Lis1 more efficiently. We further demonstrated that a Nudel mutant incapable of binding to Lis1 impaired the poleward movement of dynein and hence the dynein-mediated transport of kinetochore proteins to spindle poles along microtubules, a process contributing to inactivation of the spindle checkpoint in mitosis. These results point to the importance of Nudel-Lis1 interaction for the dynein activity in M phase and to a possible role of Nudel phosphorylation as facilitating such interaction. In addition, comparative studies suggest that NudE is also functionally related to its paralog, Nudel.