4-Hydroxytamoxifen and dexamethasone up-regulate the expression of p27 by down-regulating phosphorylation of 4E-BP1 and this down-regulation is likely to be mediated by upstream Akt/AMPK/mTOR protein kinase signaling pathways. Retinoic acids also up-regulate the expression of p27 but they do so without using any of these pathways. (a) and (b): Schematic drawings of the four upstream molecular signaling pathways of p27 expression identified in our previous study . These pathways are: pathway #1 (Figures 5a and 5b), pathway #2 (Figures 5a and 5b), pathway #3 (Figure 5b) and pathway #4 (Figure 5b). The specific inhibitors and activators used previously to identify these four pathways are indicated next to each of the four pathways. From (c) to (f): Estrogen receptor (ER) -negative MDA-MB-231 human breast cancer cells in vitro were exposed to vehicle, tamoxifen (1 μM), 4-hydroxytamoxifen (1 μM), dexamethasone (1 μM), all-trans-retinoic acid (atRA) (1 μM) or 9-cis-retinoic acid (9cRA) (1 μM) for 24 hours. Western immunoblot assays of the cells exposed to these anti-cancer agents were performed using antibodies against (c) total 4E-BP1, (d) 4E-BP1 phosphorylated at Ser65, (e) total AMPK, and (f) AMPK phosphorylated at Thr172. All assays were performed in triplicates and repeated three times. Abbreviations: RTK, receptor tyrosine kinase; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; AMPK, 5'-AMP-activated protein kinase; TSC, tuberous sclerosis complex; mTOR, mammalian target of rapamycin; eIF4E, eukaryotic translation initiation factor 4E; 4E-BP1, eIF4E-binding protein 1; MAPK, mitogen-activated protein kinase; Raf, MAP kinase kinase kinase; MEK, MAP kinase kinase; MKK, MAP kinase kinase; MNK, MAP kinase-interacting kinase; eIF2α, eukaryotic translation initiation factor 2α.