"Nutritional and chemopreventive anti-cancer agents up-regulate expression of p27Kip1, a cyclin-dependent kinase inhibitor, in mouse JB6 epidermal and human MCF7, MDA-MB-321 and AU565 breast cancer cells"

Background p27(Kip1) is a cyclin-dependent kinase inhibitor. When up-regulated, p27 inhibits G1-to-S phase transition of the cell cycle. This report addresses the question of whether various nutritional and chemopreventive anti-cancer agents up-regulate the expression of p27 in preneoplastic and neoplastic cells. Results Experimental evidence presented in the first half of this report shows that these agents fairly faithfully up-regulate expression of p27 in mouse epidermal (JB6) and human breast cancer (MCF7, MDA-MB-321, and AU565) cells. Up-regulation appears to be specific to p27 because expression of cyclin D1, E, and A, and p21Cip1/Waf1 was not modulated by these agents. Up-regulation of the expression of p27 is likely due to the activation of translation rather than transcription of p27 because (a) up-regulation is mediated by the 5'-untranslated region (-575) of the p27 gene and (b) the antibiotic actinomycin D, an inhibitor of transcription, did not attenuate the up-regulation of p27. This latter finding is likely to preclude the existence of cryptic transcription factor binding site(s) in the 5'-untranslated region of p27 gene. The experimental evidence, presented in the second half of this report, was obtained using the 5'-untranslated region (-575) of p27 gene. The evidence suggests that cancer preventive agents up-regulate expression of p27 by at least four different molecular signaling pathways: (a) Caloric restriction is likely to up-regulate p27 expression via 5'-AMP-activated protein kinase (AMPK; a metabolic energy sensor or cellular fuel gauge), tuberous sclerosis complex (TSC), and mammalian target of rapamycin (mTOR). Amino acid deficiencies also up-regulate the expression of p27 using some components of this pathway. (b) 4-Hydroxytamoxifen (but not tamoxifen), genistein (but not genistin), daidzein, and probably other nutritional and chemopreventive anti-cancer agents could up-regulate expression of p27 via receptor protein tyrosine kinases (RPTKs), phosphoinositide 3-kinase (PI3K), phosphoinosite-dependent kinase (PDK), Akt/PKB and mTOR. (c) Expression of p27 could also be up-regulated via RPTKs followed by MAPKs – MEK, ERK and p38MAPK – and probably MNK. Finally, (d) global hypomethylation of 5'-m7G cap of mRNAs could also up-regulate expression of p27. Conclusion Based on these findings, we conclude that various nutritional and chemopreventive anti-cancer agents up-regulate expression of p27 in (pre)neoplastic cells.


Background
The cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is a key cell-cycle regulator of G1-to-S phase transition [1]. Transcriptional and translational control, sequestration in cyclin D1 complexes and localization all regulate p27 in G1 phase.
Preliminary studies using either N-methyl-N-nitrosourea (MNU)-induced rat breast cancer model or human breast cancer cell lines in vitro had suggested, but not proved, that nutritional and chemopreventive anti-cancer agents increase p27 protein expression. This apparent increase in p27 protein expression might have been due to either increased synthesis or decreased degradation, or a combination of both [1].
To address this question, the effects of various nutritional and chemopreventive anti-cancer agents on the activity of the proximal 5'-upstream region of p27 gene were investigated by transient transfection assay. This study provided evidence that the up-regulation of p27 protein expression is at least in part due to increased synthesis and that this increase fairly faithfully recapitulates the cancer preventive activity of nutritional and chemopreventive anti-cancer agents. Further studies were conducted to gain some insight into the molecular basis of this increase in the synthesis of p27.

Nutritional and chemopreventive anti-cancer agents upregulate the activity of proximal 5'-upstream region (-1797) of p27 gene in a manner specific to p27
Preliminary studies using in vivo model of MNU-induced rat mammary cancer and in vitro model of cultured cells had suggested -but not proved -that various nutritional and chemopreventive anti-cancer agents, including moderate dietary restriction, up-regulated the expression of p27 (Fig. 1a).
To prove or disprove this preliminary observation, each luciferase reporter vector containing proximal 5'upstream region of the cyclin D1, cyclin A, p27 (p27-Kpn I) or p21 genes (Fig. 1b) was transiently transfected into promotion-sensitive (P+) JB6 mouse epidermal cells and then treated with all-trans-retinoic acid (atRA), 9-cis-retinoic acid (9cRA), 13-cis-retinoic acid (13cRA), phorbol 12myristate 13-acetate (TPA), 1α, 25-dihydroxyvitamin D3 (calcitriol), or dexamethasone. Phorbol 12-myristate 13acetate (TPA) is not a chemopreventive anti-cancer agent; rather it is a tumor promoter. But TPA was included here to demonstrate that it could stimulate the activity of the proximal 5'-upstream region (-1745) of cyclin D1. The TPA, three retinoic acids and dexamethasone exerted spurious effects on the backbone of the empty luciferase reporters when JB6 cells were used. Therefore, a special method, as described in the Methods and Materials section, was used to correct these effects when JB6 cells were exposed to these compounds.
The proximal 5'-upstream region of cyclin A and p21 genes were not activated by any of the compounds tested ( Fig.  1d and 1f).
4-Hydroxytamoxifen -but not tamoxifen -is the ultimate cancer preventive agents. Our results showed that 4hydroxytamoxifen -but not tamoxifen -activated -1797 p27 in ER-positive MCF7 (Fig. 2a) and ER-negative MDA-MB-231 (Fig. 2b). These results indicated that (a) the activity of -1797 p27 recapitulated the differential breast cancer preventive efficacy of 4-hydroxytamoxifen and tamoxifen and that (b) the estrogen receptor (ER) was not involved in the activation of -1797 p27. In AU565 cells, both 4-hydroxytamoxifen and tamoxifen activated -1797 Nutritional and chemopreventive anti-cancer agents up-regulate the activity of the proximal 5-upstream region (-1797) of p27 gene in a manner specific to p27 Figure 1 Nutritional and chemopreventive anti-cancer agents up-regulate the activity of the proximal 5-upstream region (-1797) of p27 gene in a manner specific to p27. (a) Preliminary studies suggesting -but not proving -that various nutritional and chemopreventive anti-cancer agents, including moderate caloric/dietary restriction, up-regulate p27 expression. (b) Schematic drawing of the proximal 5'-upstream region of the genes used in this experiment: (c) -1745 cyclin D, (d) -8100 cyclin A, (e) -1797 p27 (p27-Kpn I), and (f) -2320 p21. The JB6 cells, transfected with these proximal 5'-upstream region of the genes, were exposed to all-trans-retinoic acid (atRA) (1 μM), 9-cis-retinoic acid (9cRA) (1 μM), 13-cis-retinoic acid (13cRA) (1 μM), phorbol 12-myristate 13-acetate (TPA) (16.2 nM), 1α, 25-dihydroxyvitamin D3 (calcitriol) (240 nM), or dexamethasone (1 μM) for 24 hours. All assays were performed in triplicates and the transfection of JB6 cells with each 5'-upstream region of the genes was repeated three times.   (c) p27 (Fig. 2c), suggesting that either tamoxifen was converted to 4-hydroxytamoxifen in these cells or, as the results presented below suggest, the global rate of transcription was generally reduced in these cells, which, in turn, activated normally inactive tamoxifen by some unknown mechanisms.
Similarly, genistein -but not genistin -from soybeans is the ultimate cancer preventive agents. Our results showed that genistein -but not genistin -activated -1797 p27 in MCF7 (Fig. 2a) and MDA-MB-231 cells (Fig. 2b). These results again indicated that (a) the activity of -1797 p27 recapitulated the differential breast cancer preventive efficacy of genistein and genistin and that (b) the estrogen receptor (ER) was not involved in the activation of -1797 p27. In AU565 cells (Fig. 2c), however, both genistein and genistin activated -1797 p27 suggesting again that either genistin was converted to genistein in AU565 cells or, as the results presented below suggest, the global rate of transcription was reduced in these cells, which, in turn, activated normally inactive genistin by some unknown mechanisms.
Of the three different forms of retinoic acid tested, 9-cisretinoic acid (9cRA) most strongly activated -1797 p27, followed by all-trans-retinoic acid (atRA) and 13-cis-retinoic acid (13cRA) in all three human breast cancer cell lines. In JB6 mouse epidermal cells, these retinoic acids almost equally activated -1797 p27 (Fig. 1e). These results are compatible with those obtained using in vivo experimental animal models of breast cancer.
The estrogen receptor (ER)-negative AU565 cells were unusual in that sixteen of the eighteen compounds tested activated -1797 p27 (Fig. 2c); only mifepristone (RU486) and 1α, 25-dihydroxyvitamin D3 (calcitriol) did not activate it. This unusually high cancer preventive activity of nutritional and chemopreventive anti-cancer agents in AU565 cells might be due to its potentially reduced rate of transcription.
In summary, with the possible exception of AU565 cells, (a) activity of -1797 p27 in either estrogen receptor (ER)positive or -negative human breast cancer cells fairly faithfully recapitulated the breast cancer preventive activity in vivo of the various nutritional and chemopreventive anticancer agents and (b) the effective doses for the activation of -1797 p27 by these agents were within the range that had been found effective for in vivo rat models of breast cancer.
The results of deletion analysis suggested that, in all cases without exception ( Fig. 3b to 3e), including those not shown here, the various nutritional and chemopreventive anti-cancer agents activated proximal 5'-upstream region (-1797) of p27 gene at least through -575 p27 (5'-untranslated region (5'UTR) of p27 gene). When the regions shorter than -575 p27 (p27-5'UTR) were tested, the activities tended to either stay constant or be reduced; the activities never increased above that of -575 p27 (p27-5'UTR).

The -575 p27 (5'-untranslated region (5'UTR) of p27 gene) is unlikely to contain any cryptic transcription factor binding sites
To investigate if -575 p27 (p27-5'UTR) contains any cryptic transcription factor binding sites, the activity of the region was stimulated with 4-hydroxytamoxifen in the presence of an antibiotic actinomycin D. Figure 4a shows the schematic drawing of the pGL3 control-p27-5'UTRluciferase reporter plasmid. This plasmid -pGL3 control -contains SV40 promoter in its backbone. Preliminary tests using pGL3 control without p27-5'UTR insert had demonstrated that 4-hydroxytamoxifen, tamoxifen, or vehicle (DMSO) did not exert any spurious effects on the SV40 promoter when human breast cancer cells were used. The outline of the protocol for actinomycin D experiment is shown in Figure 4b. Actinomycin D was added one hour before the addition of 4-hydroxytamoxifen, tamoxifen, or vehicle (DMSO) and then the cells were kept exposed to both compounds for another 24 hours.
The results ( Despite this decrease in the baseline activity in the presence of actinomycin D, 4-hydroxytamoxifen still significantly up-regulated the activity of -575 p27 (p27-5'UTR) above that of vehicle (DMSO). These findings suggested that transcriptional mechanisms were not involved in the up-regulation of the activity of -575 p27 (p27-5'UTR) by 4-hydroxytamoxifen, precluding the involvement of any cryptic transcription binding sites in this region. What was more surprising was the finding that tamoxifen, which had previously been inactive in the absence of actinomycin D, significantly up-regulated the activity of -575 p27 (p27-5'UTR) in the presence of actinomycin D, suggesting that the overall level of global transcriptional rate might somehow modulate the effects of tamoxifen on the activity of -575 p27 (p27-5'UTR) in MDA-MB-231 cells.

Inhibition of certain receptor protein tyrosine kinases (RPTKs) up-regulates the activity of -575 p27 (5'untranslated region (5'UTR) of p27 gene)
The results presented above suggested that the estrogen receptor was not involved in the activation of -1797 p27 (p27-Kpn I) by 4-hydroxytamoxifen, genistein, daidzein and probably other nutritional and chemopreventive anti-cancer agents. Tamoxifen and genistein have been known to exhibit anti-estrogenic activity, but in addition, they have been reported to inhibit receptor protein tyrosine kinase (RPTK) activity at a slightly higher concentra-tions [9,10]. Epigallocatechin-3-gallate has also been reported to block activation of RPTKs such as epidermal growth factor receptor (EGFR) and HER-2/neu receptor, which are generally overexpressed or constitutively active in many human malignancies [11,12].
Although multiple RPTKs are known to be expressed in human breast cancer cells, synthetic inhibitors of four RPTKs -epidermal growth factor receptor (EGFR), HER/ ErbB, platelet-derived growth factor receptor (PDGFR) and insulin receptors (IR and IGF-1R) -were used to investigate whether they up-regulate the activity of -575 p27 (p27-5'UTR). Preliminary studies had demonstrated again that none of them exerted any spurious effects on the backbone of the empty luciferase reporter plasmids in all four types of cells used in this experiment.
AG30, a specific inhibitor of c-ErbB, did not up-regulate the activity of -575 p27 (p27-5'UTR) in any of the cells tested ( Fig. 5a to 5d).
Taken together, these results suggested that inhibition of certain RPTKs on the cell surface could up-regulate the activity of -575 p27 (p27-5'UTR).

Inhibition of certain mitogen-activated protein kinases (MAPKs) up-regulates the activity of -575 p27 (5'untranslated region (5'UTR) of p27 gene)
When the cell-surface RPTKs are inhibited, the inhibitory signal could be transmitted to the interior of the cells in two ways. The inhibition of mitogen-activated protein kinases (MAPKs) is one of them. Therefore, inhibitors of It is unlikely that -575 p27 (5'-untranslated region (5'UTR) of p27 gene) contains any cryptic transcription factor binding sites  [29] was added to the medium. One hour after the addition of these compounds, the cells were exposed to vehicle, tamoxifen (1.076 μM) or 4-hydroxytamoxifen (1.032 μM) for 24 hours. All assays were performed in triplicates and the transfection experiments were repeated three times.   MAPKs were used to investigate whether any one or more of them could up-regulate the activity of -575 p27 (p27-5'UTR). Again, none of them exerted any spurious effects on the backbone of the empty luciferase reporter plasmids in all four types of cells used in this experiment.

Inhibition of phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) also up-regulates the activity of -575 p27 (5'-untranslated region (5'UTR) of p27 gene)
The signal that the cell-surface RPTKs are inhibited is also transmitted to the interior of the cells by phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. Therefore, inhibitors of these three protein kinases were used next to investigate whether they also up-regulate the activity of -575 p27 (p27-5'UTR) in MDA-MB-231 cells. Again, preliminary experiments had been conducted to verify that none of them had exerted any spurious effects on the backbone of the empty luciferase reporter plasmids in MDA-MB-231 cells.
Also shown in Figure 7 are the effects of two cyclooxygenase inhibitors -COX-1 inhibitor FR122047 and COX-2 inhibitor II -both of which failed to up-regulate the activity of -575 p27 (p27-5'UTR) in MDA-MB-231 cells.

Inhibition of the global methylation of 5'-m 7 G-cap of mRNAs also up-regulates the activity of -575 p27 (5'untranslated region (5'UTR) of p27 gene)
There are two ways to suppress the global cap-dependent translation initiation of 5'-m 7 G-capped mRNAs, thereby potentially up-regulating the activity of -575 p27 (p27-5'UTR) by a set of mechanisms called cap-independent translation initiation. One way is to inhibit the methylation of 5'-m 7 G cap of mRNAs by S-(5'-adenosyl)-Lmethionine (AdoMet or SAM). Another way is to stimulate the inhibitory effect of endoplasmic reticulum stress by increasing the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). The results (Fig. 7) indicated that NSC 119889, a cell-permeable, competitive inhibitor of AdoMet (SAM) and which also acts as a global inhibitor of cap-dependent translation initiation, up-regulated the activity of -575 p27 (p27-5'UTR) in estrogen receptor (ER)-negative MDA-MB-231 cells. The salubrinal, a cell-permeable thiourea compound, that acts as a selective inhibitor of eukaryotic translation initiation factor 2α (eIF2α) dephosphorylation by phosphatase complex, failed to up-regulate the activity of -575 p27 (p27-5'UTR) in MDA-MB-231 cells (Fig. 7). Again, both inhibitors had not exerted any spurious effects on the backbone of the empty luciferase reporter plasmids in MDA-MB-231.

Modulation of the phosphorylation of 5'-AMP-activated protein kinase (AMPK; a metabolic energy sensor or cellular fuel gauge) either up-regulates or down-regulates the activity of -575 p27 (p27-5'UTR)
5'-AMP-activated protein kinase, known as AMPK, acts as a metabolic energy sensor or cellular fuel gauge playing a key role in the regulation of energy metabolism [13]. A number of physiological and pathophysiological stimuli, including caloric restriction, lead to an increase in the AMP: ATP ratio within the cell, which activates AMPK by phosphorylating it at Thr172 in the activation loop. When activated, AMPK modulates tuberous sclerosis complex (TSC), down-regulates phosphorylation of mammalian target of rapamycin (mTOR), eukaryotic translation initiation 4E binding protein 1 (4EBP1) and p70 S6 kinase (S6K) [13,14,30]. Thus, caloric restriction -by way of AMPK, TSC, mTOR, and 4EBP1 -might potentially upregulate the activity of -575 p27 (p27-5'UTR).
The Figure 8 shows (also see a recent report [35]). The results shown in Figure  9 also demonstrated that the removal of D-(+)-glucose from the cell culture medium up-regulated the activity of -575 p27 (p27-5'UTR). In contrast, the compounds that are known to decrease phosphorylation of AMPK -excess D-(+)-glucose and compound C -down-regulated the activity of -575 p27 (p27-5'UTR).
Metformin is a widely used drug for treatment of type 2 diabetes with no defined cellular mechanism of action. It has been suggested recently that metformin could increase phosphorylation of AMPK [15,38,39]. However, the results, as shown in Figure 8, indicated that metformin did not up-regulate the activity of -575 p27 (p27-5'UTR) in MDA-MB-231 cells.

Amino acid deficiency up-regulates the activity of -575 p27 (p27-5'UTR)
In addition to caloric restriction and growth factor signals, tuberous sclerosis complex (TSC) could transmit amino acid deficiency signals to regulate mTOR activity [13]. We already demonstrated that rapamycin, an inhibitor of mTOR phosphorylation, up-regulated the activity of -575 p27 (p27-5'UTR) in MDA-MB-231 cells (see Fig. 7). Therefore, the effects of amino acid deficiency on the activity of -575 p27 (p27-5'UTR) were investigated using MDA-MB-231 cells. Again, none of the amino acid deficiencies tested had exerted any spurious effects on the backbone of the empty luciferase reporter plasmids in MDA-MB-231 cells. As shown in Figure 9, removal of L-leucine, Lmethionine, L-cysteine, or combination of L-methionine and L-cysteine, all up-regulated the activity of -575 p27 (p27-5'UTR) in MDA-MB-231 cells. The findings of Lmethionine deficiency are interesting because L-methionine deficiency could up-regulate the activity of -575 p27 (p27-5'UTR) in two ways: one is to decrease methylation of 5'-m 7 G cap of mRNAs and another is to decrease phosphorylation of mTOR by TSC.

Discussion
The results of the study presented above indicated that various nutritional and chemopreventive anti-cancer agents up-regulate the expression of p27 in mouse epidermal (JB6) and human breast cancer (MCF7, MDA-MB-321, and AU565) cells. Up-regulation of the expression of p27 -measured by the activity of the proximal 5'upstream region (-1797) of p27 gene (p27-Kpn I)appears to be specific to p27 because expression of cyclin D1, E, and A, and p21Cip1/Waf1, another cyclin-dependent kinase inhibitor of G1-to-S phase transition, was not affected by these agents. Furthermore, up-regulation of the activity of the proximal 5'-upsteam region (-1797) of p27 gene (p27-Kpn I) fairly faithfully recapitulated the breast cancer preventive activity of various nutritional and chemopreventive anti-cancer agents.
Deletion analysis of the proximal 5'-upstream region (-1797) of p27 gene (p27-Kpn I) indicated that the activities were maintained fairly constant -neither increased nor decreased significantly -when -1797 p27 (p27-Kpn I), -774 p27 (p27-Apa I) and -575 p27 (p27-5'UTR) were used. When the fragments shorter than -575 p27 (p27-5'UTR) were used, the activities were either maintained constant or decreased. Although transcription of the p27 gene to mRNA is likely to begin at the 5'-upstream end (-575) of the 5'-untranslated region (5'UTR) of the p27 gene, it has been claimed that there could be some cryptic transcription factor binding sites within the 5'-untranslated region (5'UTR) of the p27 gene (p27-5'UTR). The general issue here is whether the up-regulation of the activity of -575 p27 (p27-5'UTR) is due to transcriptional or translational mechanism. In the earlier literature of p27 on this issue, there used to be a general consensus that changes in p27 protein levels do not correlate with changes in the transcriptional rate of p27 gene. Rather, If we assume that the activity of 5'-untranslated region (5'UTR) of the p27 gene is up-regulated by a translational mechanism, what could be the molecular basis of this mechanism? Nearly all nucleus-encoded eukaryotic proteins are translated from their respective mRNAs by a mechanism involving recognition of the 5'-m 7 G cap of the mRNAs by eukaryotic translation initiation factor 4E (eIF4E). In quiescent cells eIF4E activity is repressed, leading to a global decline in translational rate. In contrast to the translation of global mRNAs, translation of p27 mRNA is highest during quiescence, suggesting that it escapes the general repression of translational initiation.
Amino acid and glucose deficiencies up-regulate the activity of -575 p27 (p27-5'UTR) It was shown that the 5'-untranslated region (5'UTR) of the p27 mRNA mediates cap-independent translation initiation [19] and, within the 5'UTR of p27 mRNA, a U-rich element (polypyrimidine site in the putative internal ribosome entry site (IRES)) [5,20] and upstream open reading frame (uORF) [6] were reported to be necessary for the cap-independent translation initiation of p27 mRNA.
Assuming that the activity of 5'-untranslated region (5'UTR) of p27 gene is up-regulated by translational rather than transcriptional mechanisms, further experiments were conducted, using -575 p27 (p27-5'UTR) and various putative inhibitors of signaling pathways, to gain some insight into the basic mechanisms of how various nutritional and chemopreventive anti-cancer agents could transmit their signals to activate translation initiation of p27 mRNA. The results of these experiments suggested that there are at least four signaling pathways -two of them shown in Figure 10a and all four of them in Figure  10b -that could potentially transmit the signals to activate translation initiation of p27 mRNA.

Caloric restriction and amino acid deficiencies are likely to transmit signals to activate translation initiation of p27 mRNA via 5'-AMP-activated protein kinase (AMPK, metabolic energy sensor, or cellular fuel gauge), tuberous sclerosis complex (TSC), mammalian target of rapamycin (mTOR), and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and p70 S6 kinase (AMPK/TSC/ mTOR/4EBP1 and p70 S6K)
Caloric restriction could up-regulate translation initiation of p27 mRNA through its 5'-untranslated region (p27-5'UTR) by sending the signal to endoplasmic reticulum via AMPK, TSC, mTOR, and 4EBP/S6K. Amino acid deficiencies could also send the signals to endoplasmic reticulum via TSC, mTOR, and 4EBP/S6K. Deficiency of Lmethionine is an interesting case because it could also upregulate the cap-independent translation initiation of p27 mRNA by down-regulating global methylation of the 5'm 7 G-cap of other mRNAs.
Caloric restriction has long been known to activate AMPK.
The AMPK system is controlled by the balance between ATP consumption (e.g., by biosynthesis, cell growth, or muscle contraction) and ATP production via catabolism [21,22]. If the rate of ATP consumption exceeds its rate of production, such as during caloric restriction, ADP will tend to rise and be converted to AMP by the enzyme adenylate kinase. The rise in level of the activating ligand AMP, coupled with the fall in level of the inhibitory nucleotide ATP, activates AMPK, which then switches off ATPconsuming processes and switches on catabolism in an attempt to redress the balance.
4EBP1 acts as a translational repressor by binding and inhibiting the eIF4E, which recognizes the 5'-end m 7 G cap of eukaryotic mRNAs. Phosphorylation of 4EBP1 by mTOR results in a dissociation of 4EBP1 from eIF4E, thereby relieving the inhibition of 4EBP1 on eIF4Edependent (cap-dependent) translation initiation [30].
The inhibition of mTOR, therefore, results in decreased global cap-dependent translation initiation of 5'-m 7 Gcapped mRNA, but it could also increase cap-independent translation initiation of p27 mRNA through its 5'UTR.

Nutritional and chemopreventive anti-cancer agents could also up-regulate translation of p27 by transmitting inhibitory signals to receptor protein kinases (RPTKs) followed by (a) phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway, (b) MAPK/MNK signaling pathway or (c) a combination of both
Following growth factor activation of RPTKs, phosphoinositide 3-kinase (PI3K) is recruited to the receptor and activated resulting in the production of phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) [23]. This recruits Akt/ PKB to the membrane where it is phosphorylated by phosphoinositide-dependent kinase 1 (PDK1). Akt/PKB is then released from the membrane and translocate to other subcellular compartments.
Akt/PKB is involved in mTOR activation by phosphorylating mTOR at Ser2448 [13,24]. It is not yet settled whether Akt/PKB activates mTOR directly or indirectly, but recent biochemical studies indicated that Akt/PKB directly phosphorylates TSC2 and inhibits its function [13]. TSC2 inactivation by Akt/PKB may also inhibit mTOR indirectly through inhibition of the small GTPase, Rheb [13].
The inhibition of RPTKs, therefore, leads to reduction of the phosphorylation of mTOR and 4EBP/S6K, thereby attenuating the global cap-dependent translation initiation of 5'-m 7 G-capped mRNAs, but at the same time activating the cap-independent translation initiation of p27 mRNA through its 5'UTR.
Schematic drawings of the hypothetical signaling pathways that could lead to activation of the unusually long 5'-untranslated region (-575) of p27 mRNA The inhibitory signals originating from RPTKs could also be transmitted to MAPK/MNK signaling pathway. It is known that the activity of eIF4E is regulated not only by interaction with 4EBP1 but also phosphorylation by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) on Ser209. The phosphorylation of eIF4E via MNK is mediated by the activation of either the ERK or p38 pathway [25]. The results presented above indicated that the inhibition of the MEK (an upstream MAPK of ERK), ERK or p38MAPK could also decrease the phosphorylation of eIF4E, thereby reducing the global cap-dependent translation initiation of 5'-m 7 G-capped mRNAs, but at the same time activating the cap-independent translation initiation of p27 mRNA through its 5'UTR.

Global hypomethylation of the 5'-m 7 G cap of other mRNAs could also transmit signals to activate capindependent translation initiation of p27 mRNA through its 5'UTR
Nearly all mRNAs are post-transcriptionally modified at their 5' and 3' ends, by capping and polyadenylation, respectively [26][27][28]. The m 7 G-capping at their 5' end protects the nascent pre-mRNAs against degradation and failure to cap or loss of cap leads to rapid breakdown of the mRNAs. The mRNA cap (guanine-N7) methyltransferase catalyzes methyl transfer from S-adenosylmethionine (AdoMet or SAM) to GpppRNA to form m 7 GpppRNA.
The results presented above indicated that the NSC 119889, a cell-permeable, competitive inhibitor of AdoMet (SAM), inhibits global cap-dependent translation initiation of 5'-m 7 G-capped mRNAs, but it could also increase cap-independent translation initiation of p27 mRNA through its 5'UTR. This finding suggests that the epigenetic methylation hypothesis of cancer should be based not only on DNA methylation but also on mRNA methylation.
Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) is a well-documented mechanism of down-regulating protein synthesis under a variety of stress conditions, but at the same time it could also up-regulate the cap-independent translation initiation of p27 mRNA through its 5'UTR. However, contrary to this expectation, the results presented above indicated that salubrinal, a cell-permeable thiourea compound, that acts as a selective inhibitor of translation initiation factor 2α (eIF2α) dephosphorylation by phosphatase complex, failed to up-regulate the activity of 5'-untranslated region of p27 gene.

Conclusion
Based on the results presented above, we conclude that various nutritional and chemopreventive anti-cancer agents up-regulate expression of p27 in preneoplastic and neoplastic cells, thereby inhibiting G1-to-S phase transition of these cells. (a) These agents appear to up-regulate expression of p27 specifically. (b) Up-regulation of p27 fairly faithfully recapitulates cancer preventive activity of nutritional and chemopreventive anti-cancer agents. (c) Up-regulation of p27 is likely due to the activation of translation rather than transcription of p27.