Rapamycin Response in Tumorigenic and Non-Tumorigenic Hepatic Cell Lines

Abstract

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.

Impaired Phosphorylation and Ubiquitination by p70 S6 Kinase (p70S6K) and Smad Ubiquitination Regulatory Factor 1 (Smurf1) Promote Tribbles Homolog 2 (TRIB2) Stability and Carcinogenic Property in Liver Cancer

“…Inhibition of miR-17-92 or delivery of tumor suppressor microRNAs restored sensitivity to rapamycin. By profiling a total of 13 hepatic cell lines for rapamycin-induced growth inhibition, Jimenez et al (29) described that the locus of rapamycin resistance was downstream from FIGURE 12. p70S6K promoted Smurf1-induced degradation of TRIB2 via TDD, and both Smurf1 and p70S6K were critical for protein stability of TRIB2. A, rapamycin induced up-regulation of TRIB2 via TDD.…”

The physiology and pathophysiology of rapamycin resistance

“…The sensitivity of the cells to rapamycin did not correspond to individual cell lines' degree of transformation, nor was there a correlation with the ability of the drug to inhibit protein synthesis, as determined by radio-labeled leucine incorporation into protein. 33 Of note, the WB-F344 cell line, while highly sensitive to the anti-proliferative effect of rapamycin, showed no effect of rapamycin on 3 H-leucine incorporation. The H4IIE and WB-F344 cells were highly sensitive to rapamycin.…”

“…As was the case for fetal and adult liver, rapamycin potently tumorigenicity. 33 We studied two nontumorigenic hepatic cell lines, H4IIE and WB-F344. The former, derived in 1964 from a rat Reuber hepatoma, 34 has characteristics consistent with a well differentiated hepatocyte phenotype.…”

“…Roughly equivalent numbers of genes were upregulated and downregulated in response to rapamycin (60 downregulated, 66 upregulated), a finding that recapitulated our earlier results in hepatic cell lines. 33,53 In order to characterize the effects of rapamycin on gene expression in fetal liver, results were analyzed using two separate instruments, Gene Set Enrichment Analysis (GSEA), 57,58 and Ingenuity Pathways Analysis (IPA; Ingenuity ® Systems, www.ingenuity.com). Results using GSEA did not disclose a significant overrepresentation of any transcription factor binding domains.…”

The impact of age on oncogenic potential: tumor-initiating cells and the brain microenvironment

“…This kinase also promotes proliferative activity through activation of cyclin E; inhibition of mTOR with rapamycin in NPCs induces a quiescence that is reversible upon EGF treatment (Paliouras et al ., 2012). Studies have shown that nontumorigenic cell lines slow proliferation upon treatment with rapamycin, while cell lines from rat liver carcinomas are insensitive to the growth-inhibitory effects of this drug (Jimenez et al ., 2009). Although the NPCs in the aged brain remain sensitive to both the mTOR-mediated pro-proliferative effects of EGF and the growth-inhibitory effects of rapamycin (Paliouras et al ., 2012), we have observed that aged NPCs with higher rates of cell cycle progression have increased mTOR expression (unpublished observation).…”