Upstream molecular signaling pathways of p27(Kip1) expression in human breast cancer cells in vitro: differential effects of 4-hydroxytamoxifen and deficiency of either D-(+)-glucose or L-leucine

Abstract

Background: The objective of this study was to investigate whether the levels of glucose or certain amino acids could regulate the expression of a cell cycle repressor protein p27(Kip1), thereby dictating the risk of cancer in either obesity or caloric/dietary restriction. Previously, we identified and reported four different upstream molecular signaling pathways of p27 expression in human breast cancer cells. We called these four pathways as pathway #1, #2, #3 and #4. We found that 4-hydroxytamoxifen - but not tamoxifen - up-regulated the expression of p27 using pathway #1 which consisted mainly of receptor tyrosine kinases and mTORC1. We now investigate, using 4-hydroxytamoxifen as a reference anti-cancer agents, whether (a) the moderate increase in the concentration of D-(+)-glucose could down-regulate and, conversely, (b) the deficiency of D-(+)-glucose or certain L-amino acids could up-regulate the expression of p27 in these cells using pathway #2 which consists mainly of AMPK and mTORC1.

Results: Using human MDA-MB-231 breast cancer cells in vitro, these hypotheses were tested experimentally by performing p27-luciferase reporter transfection assays and western immunoblot analyses. The results obtained are consistent with these hypotheses. Furthermore, the results indicated that, although 4-hydroxytamoxifen used primarily pathway #1 to down-regulate the phosphorylation of 4E-BP1 and up-regulate the expression of p27, it also secondarily down-regulated the phosphorylation of S6K1. In contrast, the deficiency of D-(+)-glucose or L-leucine used primarily pathway #2 to down-regulate the phosphorylation of S6K1, but they also secondarily down-regulated the phosphorylation of 4E-BP1 and up-regulated the expression of p27. Finally, deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A and SIRT3.

Conclusions: (a) 4-Hydroxitamoxifen used primarily pathway #1 to up-regulate the expression of p27. (b) Moderate increase in the concentration of D-(+)-glucose used primarily pathway #2 to down-regulate the expression of p27. (c) Deficiency of D-(+)-glucose or L-leucine also used primarily pathway #2 to up-regulate the expression of p27. (d) Deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A in the Complex V of respiratory oxidation-phosphorylation chain and mitochondrial SIRT3. The SIRT3 is one of the seven mammalian anti-aging as well as anti-metabolic sirtuins.

Figure 1

4-Hydroxytamoxifen - but not tamoxifen - up-regulated the expression of p27 in estrogen receptor (ER) and LKB1-double negative human MDA-MB-231 breast cancer cells in vitro. (a) Schematic drawing of the luciferase reporter plasmids containing the following proximal 5'-upstream regions of the p27 gene, namely -1797 p27 (p27-Kpn I), -774 p27 (p27-Apa I), and -575 p27 (p27-5'UTR). (b) Tamoxifen did not up-regulate the relative luciferase activity of p27-Kpn 1, Apa I or 5'UTR in MDA-MB-231 cells (c) 4-Hydroxytamoxifen up-regulated the relative luciferase activity of the three p27- luciferase reporter plasmids in MDA-MB-231 cells. In all experiments, the cells were exposed to either DMSO or 1 μM each of tamoxifen or 4-hydroxytamoxifen for 24 hours. All assays were performed in triplicates and repeated three times. (d) Schematic diagram showing how 4-hydroxytamoxifen could up-regulate the expression of p27, arrest the progression of cell cycle from G1 to S phase, and inhibit DNA replication.

Figure 2

Moderate increase in the concentration of D-(+)-glucose down-regulated, but deficiency of D-(+)-glucose, L-leucine, L-methionine, L-cysteine or combination of L-methionine and L-cysteine up-regulated the relative luciferase activity of p27-5'UTR in human MDA-MB-231 breast cancer cells in vitro. (a) Schematic drawing of the luciferase reporter plasmid containing a proximal 5'-upstream region (-575) of the p27 gene (p27-5'UTR). (b) Moderate increase in the concentration of D-(+)-glucose down-regulated the relative luciferase activity of p27-5'UTR in MDA-MB-231 cells. This panel also shows that rotenone (inhibitor of NADH dehydrogenase (Complex 1) of the mitochondrial respiratory oxidation-phosphorylation chain) and AICA riboside (inhibitor of AMPK (5'-AMP-activated protein kinase)) up-regulated the relative luciferase activity of p27-5'UTR in MDA-MB-231 cells. In contrast, compound C (activator of AMPK) down-regulated the relative luciferase activity of p27-5'UTR in these cells. Metformin (the most widely prescribed anti-diabetic drug that activates AMPK in the cells by mechanisms that are dependent on its upstream kinase, the tumor suppressor LKB1) did not either up or down-regulate the relative luciferase activity of p27-5UTR probably because MDA-MB-231 cells lack LKB1. (c) This panel shows that deficiency of D-(+)-glucose, L-leucine, L-methionine, L-cysteine, or combination of L-methionine and L-cysteine up-regulated the relative luciferase activity of p27-5'UTR in MDA-MB-231 cells. All assays were performed in triplicates and repeated three times.

Figure 3

Schematic diagram of the four different upstream molecular signaling pathways of p27 expression that could lead to either increased or decreased expression of p27 in human breast cancer cells in vitro. (a) Previously, we identified and reported four different upstream molecular signaling pathways of the expression of p27 [1,2]. We also reported previously that 4-hydroxytamoxifen - but not tamoxifen - up-regulated the expression of p27 by using pathway #1 which consists mainly of receptor tyrosine kinases (RTKs) and mTORC1 [2]. Now, we hypothesize that (i) moderate increase in the concentration of D-(+)-glucose down-regulates and (ii) deficiency of D-(+)-glucose or certain L-amino acids up-regulates the expression of p27 using pathway #2 which consists mainly of AMPK and mTORC1. (b) We also identified and reported previously two additional upstream molecular signaling pathways - namely #3 and #4 - of the expression of p27 [1,2]. (c and e) Western immunoblot analysis of the effects of D-(+)-glucose deficiency, DMSO, tamoxifen and 4-hydroxytamoxifen on the expression of p27 protein in MDA-MB-231 cells. (d and e) Western immunoblot analysis of the effects of the deficiency of L-leucine, L-methionine or L-cysteine on the expression of p27 protein in these cells. All assays were performed in triplicates and repeated three times.

Figure 4

Effects of tamoxifen, 4-hydroxytamoxifen, and deficiency of D-(+)-glucose, L-leucine, L-methionine or L-cysteine on the phosphorylation of 4E-BP1 in MDA-MB-231 cells. (a and e) Western immunoblot analysis of the effects of D-(+)-glucose deficiency, DMSO, tamoxifen and 4-hydroxytamoxifen on the expression of total 4E-BP1 (eukaryotic translation initiation factor 4E binding protein 1) and (b and e) phosphorylated 4E-BP1. (c and e) Western immunoblot analysis of the effects of the deficiency of L-leucine, L-methionine, or L-cysteine on the expression of total 4E-BP1 and (d and e) phosphorylated 4E-BP1. All assays were performed in triplicates and repeated three times. (f) Correlation between the degree of expression of p27 protein and the degree of down-regulation of the phosphorylated 4E-BP1.

Figure 5

Effects of tamoxifen, 4-hydroxytamoxifen, and deficiency of D-(+)-glucose, L-leucine, L-methionine or L-cysteine on the phosphorylation of S6K1 in MDA-MB-231 cells. (a and e) Western immunoblot analysis of the effects of D-(+)-glucose deficiency, DMSO, tamoxifen and 4-hydroxytamoxifen on the expression of total S6K1 and (b and e) phosphorylated S6K1. (c and e) Westerm immunoblot analysis of the effects of the deficiency of L-leucine, L-methionine or L-cysteine on the expression of total S6K1 and (d and e) phosphorylated S6k1. All assays were performed in triplicates and repeated three times. (f) Correlation between the degree of expression of p27 protein and the degree of down-regulation of phosphorylated S6k1.

Figure 6

Effects of tamoxifen, 4-hydroxytamoxifen and deficiency of D-(+)-glucose on the expression of HIF-1a, SREBP-1 and phosphorylation of eEF2k in MDA-MB-231 cells. (a and e) Western immunoblot analysis of the effects of D-(+)-glucose deficiency, DMSO, tamoxifen and 4-hydroxytamoxifen on the expression of HIF1α. (b and e) SREBP-1, (c and e) total eEF2k and (d and e) phosphorylated eEF2k. All assays were performed in triplicates and repeated three times.

Figure 7

Effects of tamoxifen, 4-hydroxytamoxifen and deficiency of D-(+)-glucose, L-leucine, L-methionine or L-cysteine on the expression of mitochondrial ATP5A and SIRT3 in MDA-MB-231 cells. (a and e) Western immunoblot analysis of the effects of D-(+)-glucose deficiency, DMSO, tamoxifen, 4-hydroxytamoxifen and (b and e) deficiency of L-leucine, L-methionine or L-cysteine on the expression of mitochondrial ATP5a in the Complex V of respiratory oxidation-phosphorylation system. (c and e) Western immunoblot analysis of the effects of D-(+)-glucose deficiency, DMSO, tamoxifen, 4-hydroxytamoxifen and (d and e) deficiency of L-leucine, L-methionine or L-cysteine on the expression of mitochondrial SIRT3. The SIRT3 is one of the seven mammalian anti-aging and anti-metabolic sirtuins. All assays were performed in triplicates and repeated three times.

Figure 8

Schematic diagram of the hypothetical molecular signaling pathways of the expression of p27 by which moderate increase in the concentration of D-(+)-glucose down-regulates and deficiency of D-(+)-glucose or L-leucine up-regulates the expression of p27. This summary diagram shows the outline of how 4-hydroxytamoxifen uses primarily pathway #1 to up-regulate the expression of p27, arrest the G1-to-S phase transition of cell cycle, and inhibit DNA replication in human breast cancer cells in vitro. 4-Hydroxytamoxifen preferentially phosphorylates 4E-BP1 over S6K1. The diagram also shows the outline of how the deficiency of D-(+)-glucose uses primarily pathway #2 to up-regulate the expression of p27, arrest the G1-to-S phase transition of cell cycle, and inhibit DNA replication in human breast cancer cells in vitro. D-(+)-Glucose deficiency preferentially phosphorylates S6K1 over 4E-BP1. The diagram also shows that the deficiency of L-leucine enters the pathway #2 at points different from the deficiency of D-(+)-glucose. Finally, the diagram shows that deficiency of D-(+)-glucose or L-leucine uses L-upstream molecular signaling pathway #2 of the expression of p27 to up-regulate the phosphorylation of AMPK and the expression of mitochondrial ATP5A and SIRT3. The mitochondrial SIRT3 exerts anti-aging and other metabolic effects on the cells.