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. 2009 Jan;8(1):83-93.
doi: 10.1158/1535-7163.MCT-08-0254.

Regulation of D-cyclin translation inhibition in myeloma cells treated with mammalian target of rapamycin inhibitors: rationale for combined treatment with extracellular signal-regulated kinase inhibitors and rapamycin

Patrick Frost  1 , Yijiang Shi, Bao Hoang, Joseph Gera, Alan Lichtenstein
Affiliations

Regulation of D-cyclin translation inhibition in myeloma cells treated with mammalian target of rapamycin inhibitors: rationale for combined treatment with extracellular signal-regulated kinase inhibitors and rapamycin

Patrick Frost et al. Mol Cancer Ther. 2009 Jan.

Abstract

We have shown that heightened AKT activity sensitized multiple myeloma cells to the antitumor effects of the mammalian target of rapamycin inhibitor CCI-779. To test the mechanism of the AKT regulatory role, we stably transfected U266 multiple myeloma cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analogue, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mammalian target of rapamycin inhibitors to down-regulate D-cyclin expression was significantly greater in AKT-transfected multiple myeloma cells due, in part, to the ability of AKT to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was shown in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild-type PTEN. Because extracellular signal-regulated kinase (ERK)/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells showed significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant "low-AKT" myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down-regulated D-cyclin protein expression and G1 arrest. However, ectopic overexpression of an activated MEK gene did not increase cap-independent translation of D-cyclin in "high-AKT" myeloma cells, indicating that mitogen-activated protein kinase/ERK kinase/ERK activity was required, but not sufficient, for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in multiple myeloma cells and ERK facilitates activity.

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Figures

Figure 1
Figure 1. AKT-mediates the sensitivity of U266 isogenic myeloma cells to cytostatic effects of mTOR inhibitors
(A) AKT activation in isogenic U266 cells by immunoblot assay for expression of total AKT and AKT phosphorylation at serine residue 473 and threonine 308, or total AKT levels in cells treated with or without IGF-1 (400ng/ml) for 2 hours. (B) Effects on cellular proliferation (top panel) in U266AKT (open bars) and U266EV (closed bars) and MTT assay (bottom panel) measuring sensitivity of U266AKT (open circles) and U266EV (closed circles) cells to rapamycin performed after 48-hour culture with increasing concentration of drug. Results are the means ± SD of three separate experiments. AKT-transfected cells are more sensitive (p
Figure 1
Figure 1. AKT-mediates the sensitivity of U266 isogenic myeloma cells to cytostatic effects of mTOR inhibitors
(A) AKT activation in isogenic U266 cells by immunoblot assay for expression of total AKT and AKT phosphorylation at serine residue 473 and threonine 308, or total AKT levels in cells treated with or without IGF-1 (400ng/ml) for 2 hours. (B) Effects on cellular proliferation (top panel) in U266AKT (open bars) and U266EV (closed bars) and MTT assay (bottom panel) measuring sensitivity of U266AKT (open circles) and U266EV (closed circles) cells to rapamycin performed after 48-hour culture with increasing concentration of drug. Results are the means ± SD of three separate experiments. AKT-transfected cells are more sensitive (p
Figure 2
Figure 2. Inhibition of cyclin D1 expression by mTOR inhibitors is AKT-dependent
(A) Immunoblot assay for expression of cyclin D1 or actin in extracted protein from isogenic U266 cells cultured with increasing concentrations of rapamycin for 48 hours. Densitometry was performed on equally exposed autoradiographs and data are presented as cyclin D1/actin ratios compared to untreated cells (bottom panel). This experiment was repeated 3 times with identical results. (B) Effects of CCI-779 on expression of cyclin D1. U266-challenged mice were treated with varying doses of CCI-779 (shown above blots as mg/kg) for 13 days, after which the tumor nodules were harvested and the extracted protein immunoblotted for expression of cyclin D1 and actin. Data are from pooled extract (4 mice/group) and are presented as in figure 2A. (C) Effects of rapamycin on retinoblastoma (Rb) protein. Protein extracted from U266 cells cultured with increasing concentrations of rapamycin for 48 hours was immunoblotted for total Rb, and phosphorylated Rb at residues 780 and 807/811.
Figure 2
Figure 2. Inhibition of cyclin D1 expression by mTOR inhibitors is AKT-dependent
(A) Immunoblot assay for expression of cyclin D1 or actin in extracted protein from isogenic U266 cells cultured with increasing concentrations of rapamycin for 48 hours. Densitometry was performed on equally exposed autoradiographs and data are presented as cyclin D1/actin ratios compared to untreated cells (bottom panel). This experiment was repeated 3 times with identical results. (B) Effects of CCI-779 on expression of cyclin D1. U266-challenged mice were treated with varying doses of CCI-779 (shown above blots as mg/kg) for 13 days, after which the tumor nodules were harvested and the extracted protein immunoblotted for expression of cyclin D1 and actin. Data are from pooled extract (4 mice/group) and are presented as in figure 2A. (C) Effects of rapamycin on retinoblastoma (Rb) protein. Protein extracted from U266 cells cultured with increasing concentrations of rapamycin for 48 hours was immunoblotted for total Rb, and phosphorylated Rb at residues 780 and 807/811.
Figure 2
Figure 2. Inhibition of cyclin D1 expression by mTOR inhibitors is AKT-dependent
(A) Immunoblot assay for expression of cyclin D1 or actin in extracted protein from isogenic U266 cells cultured with increasing concentrations of rapamycin for 48 hours. Densitometry was performed on equally exposed autoradiographs and data are presented as cyclin D1/actin ratios compared to untreated cells (bottom panel). This experiment was repeated 3 times with identical results. (B) Effects of CCI-779 on expression of cyclin D1. U266-challenged mice were treated with varying doses of CCI-779 (shown above blots as mg/kg) for 13 days, after which the tumor nodules were harvested and the extracted protein immunoblotted for expression of cyclin D1 and actin. Data are from pooled extract (4 mice/group) and are presented as in figure 2A. (C) Effects of rapamycin on retinoblastoma (Rb) protein. Protein extracted from U266 cells cultured with increasing concentrations of rapamycin for 48 hours was immunoblotted for total Rb, and phosphorylated Rb at residues 780 and 807/811.
Figure 3
Figure 3. Polysome analysis of isogenic U266 multiple myeloma cell lines
(A) The cells were treated with and without 10 nM rapamycin for 48 hours and polysomes were separated from monosomes by sucrose gradients. Northern blots were performed on the gradient fractions for cyclin D1 (top panels) and actin (bottom panels) mRNAs. The percent of total mRNA present (measured by densitometry of equally exposed Autoradiographs) in the polysomal fractions (fractions 4–11) are shown to the right of the autoradiographs. This experiment was performed one additional time with identical results. (B) Summary of polysome analysis of isogenic U266 multiple myeloma cell lines from two separate experiments. The percentage of cyclin mRNA associated with monosome (poorly translated) or polysome (well translated) fractions in U266 cells cultured with or without 10 nM rapamycin for 48 hours. Data represents mean ± SD. (C) Schematic diagrams of the dicistronic constructs used in this study. Constructs used are pRF, pRCD1F, which contains the 5′-UTR of human cyclin D1 in the intracistronic space, and pRp27F, which contains the 5′UTR of human p27 in the intracistronic space. Luc, luciferase. (D) U266 cell lines were transfected with the indicated constructs and treated with or without rapamycin (100nM) for 18 hours. The cells were lysed and the firefly and Renilla luciferase activity was measured. Data is shown as firefly/Renilla luciferase ratio of U266EV (white boxes) and U266AKT cells (black boxes) transfected with the indicated constructs.
Figure 3
Figure 3. Polysome analysis of isogenic U266 multiple myeloma cell lines
(A) The cells were treated with and without 10 nM rapamycin for 48 hours and polysomes were separated from monosomes by sucrose gradients. Northern blots were performed on the gradient fractions for cyclin D1 (top panels) and actin (bottom panels) mRNAs. The percent of total mRNA present (measured by densitometry of equally exposed Autoradiographs) in the polysomal fractions (fractions 4–11) are shown to the right of the autoradiographs. This experiment was performed one additional time with identical results. (B) Summary of polysome analysis of isogenic U266 multiple myeloma cell lines from two separate experiments. The percentage of cyclin mRNA associated with monosome (poorly translated) or polysome (well translated) fractions in U266 cells cultured with or without 10 nM rapamycin for 48 hours. Data represents mean ± SD. (C) Schematic diagrams of the dicistronic constructs used in this study. Constructs used are pRF, pRCD1F, which contains the 5′-UTR of human cyclin D1 in the intracistronic space, and pRp27F, which contains the 5′UTR of human p27 in the intracistronic space. Luc, luciferase. (D) U266 cell lines were transfected with the indicated constructs and treated with or without rapamycin (100nM) for 18 hours. The cells were lysed and the firefly and Renilla luciferase activity was measured. Data is shown as firefly/Renilla luciferase ratio of U266EV (white boxes) and U266AKT cells (black boxes) transfected with the indicated constructs.
Figure 4
Figure 4. Inhibition of AKT activity confers resistance to rapamycin in OPM-2 cells
(A) Immunoblot assay for expression of PTEN, total and phospho-AKT (ser473) in extracted protein from OPM-2 cells infected with PTEN-adenovirus or control-adenovirus. OPM-2 cells were transduced with control (EV) or wild-type (PTEN) adenovirus for 2 hours. The adenovirus was then washed away, and the cells were resuspended in medium for an additional 24 hours. (B) Immunoblot assay for expression of PTEN and actin (top panel) or cyclin D1 (bottom panel) in extracted protein from OPM-2 cells transduced with wild-type PTEN or control adenovirus as described above. The cells were then cultured with increasing concentrations of rapamycin for 48 hours. (C) OPM-2 cells transduced with wild-type PTEN or control adenovirus as described above were transfected by electroporation with the indicated dual luciferase reporter constructs for an additional 18 hours. The cells were lysed and the firefly and Renilla luciferase activity was measured. Data is shown as firefly/Renilla luciferase ratio of OPM-2 control (white boxes) or OPM-2 wild-type PTEN transfected cells (black boxes).
Figure 5
Figure 5. Cyclin D1 IRES activity requires ERK signaling in vivo
(A) Top panel: Western blot analysis of total and phosphorylated ERK or p38 expression in U266EV and U266AKT cells. (B) U266 cell lines were transfected with the pRCD1F construct containing the cyclin D1 IRES and treated with or without 100nM rapamycin (RAPA), 1 μM U0126 (a MEK inhibitor) or 25μM SB202190 (a p38 inhibitor) for 18 hours. The cells were lysed and the firefly and Renilla luciferase activity was measured. Data is shown as relative Renilla luciferase (white boxes) or firefly luciferase (black boxes). (C) Phosphorylated ERK protein was immunoprecipitated from lysate of isogenic U266 cells treated with indicated concentration of ERK/MEK inhibitor (U0126) with ELK-1 used as substrate for ERK kinase activity. The ERK kinase activity is shown by immunoblot assay for phosphorylated ELK-1 (pELK-1).
Figure 6
Figure 6. Inhibition of ERK activity overcomes resistance to rapamycin in U266 cells
(A) FACS analysis of hypotonic PI-stained U266EV cells treated with 1 nM rapamycin, or 1 μM U0126 MEK/ERK inhibitor, or both drugs for 48 hours. The percentage of cells in G1 (black bars) or S phase (open bars) were determined from the generated histograms using ModFit cell cycle analysis software. This experiment was repeated 3 times with similar results. *=PRenilla luciferase activity was measured as described above. Data is shown as relative Renilla luciferase (white boxes) or firefly luciferase (black boxes). * indicates P<0.05.
Figure 6
Figure 6. Inhibition of ERK activity overcomes resistance to rapamycin in U266 cells
(A) FACS analysis of hypotonic PI-stained U266EV cells treated with 1 nM rapamycin, or 1 μM U0126 MEK/ERK inhibitor, or both drugs for 48 hours. The percentage of cells in G1 (black bars) or S phase (open bars) were determined from the generated histograms using ModFit cell cycle analysis software. This experiment was repeated 3 times with similar results. *=PRenilla luciferase activity was measured as described above. Data is shown as relative Renilla luciferase (white boxes) or firefly luciferase (black boxes). * indicates P<0.05.
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