BackgroundThe mTOR inhibitor rapamycin has anti-tumor activity across a variety of human cancers, including hepatocellular carcinoma. However, resistance to its growth inhibitory effects is common. We hypothesized that hepatic cell lines with varying rapamycin responsiveness would show common characteristics accounting for resistance to the drug.Methodology/Principal FindingsWe profiled a total of 13 cell lines for rapamycin-induced growth inhibition. The non-tumorigenic rat liver epithelial cell line WB-F344 was highly sensitive while the tumorigenic WB311 cell line, originally derived from the WB-F344 line, was highly resistant. The other 11 cell lines showed a wide range of sensitivities. Rapamycin induced inhibition of cyclin E–dependent kinase activity in some cell lines, but the ability to do so did not correlate with sensitivity. Inhibition of cyclin E–dependent kinase activity was related to incorporation of p27Kip1 into cyclin E–containing complexes in some but not all cell lines. Similarly, sensitivity of global protein synthesis to rapamycin did not correlate with its anti-proliferative effect. However, rapamycin potently inhibited phosphorylation of two key substrates, ribosomal protein S6 and 4E-BP1, in all cases, indicating that the locus of rapamycin resistance was downstream from inhibition of mTOR Complex 1. Microarray analysis did not disclose a unifying mechanism for rapamycin resistance, although the glycolytic pathway was downregulated in all four cell lines studied.Conclusions/SignificanceWe conclude that the mechanisms of rapamycin resistance in hepatic cells involve alterations of signaling downstream from mTOR and that the mechanisms are highly heterogeneous, thus predicting that maintaining or promoting sensitivity will be highly challenging.
BackgroundWe investigated mTOR regulation of gene expression by studying rapamycin effect in two hepatic cell lines, the non-tumorigenic WB-F344 cells and the tumorigenic WB311 cells. The latter are resistant to the growth inhibitory effects of rapamycin, thus providing us with an opportunity to study the gene expression effects of rapamycin without confounding effects on cell proliferation.Methodology/Principal FindingsThe hepatic cells were exposed to rapamycin for 24 hr. Microarray analysis on total RNA preparations identified genes that were affected by rapamycin in both cell lines and, therefore, modulated independent of growth arrest. Further studies showed that the promoter regions of these genes included E-box-containing transcription factor binding sites at higher than expected rates. Based on this, we tested the hypothesis that c-Myc is involved in regulation of gene expression by mTOR by comparing genes altered by rapamycin in the hepatic cells and by c-Myc induction in fibroblasts engineered to express c-myc in an inducible manner. Results showed enrichment for c-Myc targets among rapamycin sensitive genes in both hepatic cell lines. However, microarray analyses on wild type and c-myc null fibroblasts showed similar rapamycin effect, with the set of rapamycin-sensitive genes being enriched for c-Myc targets in both cases.Conclusions/SignificanceThere is considerable overlap in the regulation of gene expression by mTOR and c-Myc. However, regulation of gene expression through mTOR is c-Myc-independent and cannot be attributed to the involvement of specific transcription factors regulated by the rapamycin-sensitive mTOR Complex 1.
The Ser/Thr phosphatase PP2A is a set of multisubunit enzymes that regulate many cellular processes. In yeast, the PP2A regulatory subunit Tap42 forms part of the Target of Rapamycin (TOR) signaling pathway that links nutrient and energy availability to cell growth. The physiological intersection between the mammalian orthologs of Tap42 and TOR, α4 and mTOR, has not been fully characterized. We used two in vivo models of liver growth in the rat, late gestation fetal development and regeneration after partial hepatectomy, to explore the regulation of the α4-containing form of PP2A. The α4/PP2A catalytic subunit (α4/PP2A-C) complex was present in both fetal and adult liver extracts. There was a trend towards higher levels of α4 protein in fetal liver, but the complex was more abundant in adult liver. Fractionation of extracts by ion exchange chromatography and transient transfection of the AML12 mouse hepatic cell line indicated that α4 associates with PP2A-C but that these complexes have low catalytic activity with both peptide and protein substrates. α4 was able to associate with forms of PP2A-C that were both methylated and non-methylated at the carboxy-terminus. The mTOR inhibitor rapamycin did not block the formation of α4/PP2A-C in liver or hepatic cells, nor did it appear to modulate PP2A activity. Furthermore, sensitivity to the growth inhibitory effects of rapamycin among a panel of hepatic cell lines did not correlate with levels of α4 or α4/PP2A-C. Our results indicate that the yeast Tap42/TOR paradigm is not conserved in hepatic cells. KeywordsProtein phosphorylation; mTOR; rapamycin; regeneration; cell growth PP2A is a major Ser/Thr phosphatase that plays a significant role in the regulation of many cellular processes, including metabolism, transcription, translation, cell cycle progression, cell growth and apoptosis [Janssens and Goris, 2001;Mumby and Walter, 1993;Sontag, 2001;Zolnierowicz, 2000]. The activity of such a versatile enzyme must therefore be tightly controlled in vivo. Indeed, the catalytic subunit of PP2A (PP2A-C) is regulated at many levels. The C-terminus of PP2A-C is post-translationally modified by phosphorylation at Y307 and methyl esterification at L309. Phosphorylation results in inhibition of PP2A activity [Chen et al., 1992;Guo and Damuni, 1993] while methylation is thought to play an important function in regulatory subunit binding [Bryant et al., 1999;Tolstykh et al., 2000;Wu et al., 2000;Yu et al., 2001]. More specifically, PP2A-C methylation can influence the association of PP2A-C with specific regulatory subunits, thereby serving to determine the NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript composition, subcellular localization and function of PP2A-C holoenzymes in a variety of cell types [Gentry et al., 2005;Longin et al., 2007;Nunbhakdi-Craig et al., 2007].The prototypical PP2A enzyme is a heterodimer that consists of the catalytic C subunit and scaffolding A subunit. The substrate specificity and subcellular localization of PP2A are...
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