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Synergistic growth inhibition by Iressa and Rapamycin is modulated by VHL mutations in renal cell carcinoma.

Gemmill RM, Zhou M, Costa L, Korch C, Bukowski RM, Drabkin HA - Br. J. Cancer (2005)

Bottom Line: We found that RCC cell lines expressed EGFR mRNA in the near-absence of other ErbB family members.Primary RCCs vs cell lines contained similar amounts of phospho-ERK1/2, much higher levels of ErbB-3, less phospho-AKT, and no evidence of phospho-RPS6, suggesting that mTOR activity was reduced.However, apparent differences between primary tumours and cell lines require further investigation.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Oncology, University of Colorado at Denver and Health Sciences and Cancer Centers, Mail Stop 8117, PO Box 6511, Aurora, CO 80045-0511, USA. robert.gemmill@uchsc.edu

ABSTRACT
Epidermal growth factor receptor (EGFR) and tumour growth factor alpha (TGFalpha) are frequently overexpressed in renal cell carcinoma (RCC) yet responses to single-agent EGFR inhibitors are uncommon. Although von Hippel-Lindau (VHL) mutations are predominant, RCC also develops in individuals with tuberous sclerosis (TSC). Tuberous sclerosis mutations activate mammalian target of rapamycin (mTOR) and biochemically resemble VHL alterations. We found that RCC cell lines expressed EGFR mRNA in the near-absence of other ErbB family members. Combined EGFR and mTOR inhibition synergistically impaired growth in a VHL-dependent manner. Iressa blocked ERK1/2 phosphorylation specifically in wt-VHL cells, whereas rapamycin inhibited phospho-RPS6 and 4E-BP1 irrespective of VHL. In contrast, phospho-AKT was resistant to these agents and MYC translation initiation (polysome binding) was similarly unaffected unless AKT was inhibited. Primary RCCs vs cell lines contained similar amounts of phospho-ERK1/2, much higher levels of ErbB-3, less phospho-AKT, and no evidence of phospho-RPS6, suggesting that mTOR activity was reduced. A subset of tumours and cell lines expressed elevated eIF4E in the absence of upstream activation. Despite similar amounts of EGFR mRNA, cell lines (vs tumours) overexpressed EGFR protein. In the paired cell lines, PRC3 and WT8, EGFR protein was elevated post-transcriptionally in the VHL mutant and EGF-stimulated phosphorylation was prolonged. We propose that combined EGFR and mTOR inhibitors may be useful in the subset of RCCs with wt-VHL. However, apparent differences between primary tumours and cell lines require further investigation.

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Growth inhibition by Iressa and rapamycin in derivatives of 786-O cells. The parental RCC line 786-O and three stably transfected derivatives were tested for the effects of varying doses of Iressa and rapamycin on growth using MTT assays. MTT absorbance values from single and combined agents were converted to the combination index (see Methods).
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fig2: Growth inhibition by Iressa and rapamycin in derivatives of 786-O cells. The parental RCC line 786-O and three stably transfected derivatives were tested for the effects of varying doses of Iressa and rapamycin on growth using MTT assays. MTT absorbance values from single and combined agents were converted to the combination index (see Methods).

Mentions: The quantitative EGFR results suggested that a selective inhibitor, such as Iressa (Gefitinib, ZD1839), might inhibit growth especially when combined with rapamycin. We tested this hypothesis initially using the paired cell lines, WT8 and PRC3. These lines were derived from 786-O, a clear-cell carcinoma that contains a deletion and frameshift of the VHL gene (Iliopoulos et al, 1995). WT8 cells contain stably transfected, HA-tagged VHL (p24 form), while PRC3 cells contain the vector-only control. Low-density cultures of WT8 and PRC3 cells were treated with varying concentrations of Iressa, rapamycin or the combination. After 5 days, growth was measured by MTT assays and the results converted to a combination index using CALCUSYN (Chou and Talalay, 1984). Values much less than 1.0 indicate synergy while values much greater than 1.0 are indicative of antagonism. As shown in Figure 2, Iressa plus rapamycin at various doses synergistically inhibited growth of WT8 cells. Nearly identical results were obtained using an independent stable VHL transfectant, MPR6 (Schoenfeld et al, 1998). These results include Iressa concentrations within the reported IC50 range for EGFR (i.e. 30–50 nM). At high Iressa and low rapamycin concentrations, a combination index close to 1.0 was obtained (indicating no interaction). In contrast, in the VHL-mutant cell lines 786-O and PRC3, low concentrations of Iressa plus rapamycin were antagonistic. Thus, the introduction of a wt-VHL gene into 786-O cells confers sensitivity (synergy) to the combination of EGFR plus mTOR inhibition at low drug concentrations. Of note, the levels of rapamycin used are readily achievable in patients.


Synergistic growth inhibition by Iressa and Rapamycin is modulated by VHL mutations in renal cell carcinoma.

Gemmill RM, Zhou M, Costa L, Korch C, Bukowski RM, Drabkin HA - Br. J. Cancer (2005)

Growth inhibition by Iressa and rapamycin in derivatives of 786-O cells. The parental RCC line 786-O and three stably transfected derivatives were tested for the effects of varying doses of Iressa and rapamycin on growth using MTT assays. MTT absorbance values from single and combined agents were converted to the combination index (see Methods).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2361810&req=5

fig2: Growth inhibition by Iressa and rapamycin in derivatives of 786-O cells. The parental RCC line 786-O and three stably transfected derivatives were tested for the effects of varying doses of Iressa and rapamycin on growth using MTT assays. MTT absorbance values from single and combined agents were converted to the combination index (see Methods).
Mentions: The quantitative EGFR results suggested that a selective inhibitor, such as Iressa (Gefitinib, ZD1839), might inhibit growth especially when combined with rapamycin. We tested this hypothesis initially using the paired cell lines, WT8 and PRC3. These lines were derived from 786-O, a clear-cell carcinoma that contains a deletion and frameshift of the VHL gene (Iliopoulos et al, 1995). WT8 cells contain stably transfected, HA-tagged VHL (p24 form), while PRC3 cells contain the vector-only control. Low-density cultures of WT8 and PRC3 cells were treated with varying concentrations of Iressa, rapamycin or the combination. After 5 days, growth was measured by MTT assays and the results converted to a combination index using CALCUSYN (Chou and Talalay, 1984). Values much less than 1.0 indicate synergy while values much greater than 1.0 are indicative of antagonism. As shown in Figure 2, Iressa plus rapamycin at various doses synergistically inhibited growth of WT8 cells. Nearly identical results were obtained using an independent stable VHL transfectant, MPR6 (Schoenfeld et al, 1998). These results include Iressa concentrations within the reported IC50 range for EGFR (i.e. 30–50 nM). At high Iressa and low rapamycin concentrations, a combination index close to 1.0 was obtained (indicating no interaction). In contrast, in the VHL-mutant cell lines 786-O and PRC3, low concentrations of Iressa plus rapamycin were antagonistic. Thus, the introduction of a wt-VHL gene into 786-O cells confers sensitivity (synergy) to the combination of EGFR plus mTOR inhibition at low drug concentrations. Of note, the levels of rapamycin used are readily achievable in patients.

Bottom Line: We found that RCC cell lines expressed EGFR mRNA in the near-absence of other ErbB family members.Primary RCCs vs cell lines contained similar amounts of phospho-ERK1/2, much higher levels of ErbB-3, less phospho-AKT, and no evidence of phospho-RPS6, suggesting that mTOR activity was reduced.However, apparent differences between primary tumours and cell lines require further investigation.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Oncology, University of Colorado at Denver and Health Sciences and Cancer Centers, Mail Stop 8117, PO Box 6511, Aurora, CO 80045-0511, USA. robert.gemmill@uchsc.edu

ABSTRACT
Epidermal growth factor receptor (EGFR) and tumour growth factor alpha (TGFalpha) are frequently overexpressed in renal cell carcinoma (RCC) yet responses to single-agent EGFR inhibitors are uncommon. Although von Hippel-Lindau (VHL) mutations are predominant, RCC also develops in individuals with tuberous sclerosis (TSC). Tuberous sclerosis mutations activate mammalian target of rapamycin (mTOR) and biochemically resemble VHL alterations. We found that RCC cell lines expressed EGFR mRNA in the near-absence of other ErbB family members. Combined EGFR and mTOR inhibition synergistically impaired growth in a VHL-dependent manner. Iressa blocked ERK1/2 phosphorylation specifically in wt-VHL cells, whereas rapamycin inhibited phospho-RPS6 and 4E-BP1 irrespective of VHL. In contrast, phospho-AKT was resistant to these agents and MYC translation initiation (polysome binding) was similarly unaffected unless AKT was inhibited. Primary RCCs vs cell lines contained similar amounts of phospho-ERK1/2, much higher levels of ErbB-3, less phospho-AKT, and no evidence of phospho-RPS6, suggesting that mTOR activity was reduced. A subset of tumours and cell lines expressed elevated eIF4E in the absence of upstream activation. Despite similar amounts of EGFR mRNA, cell lines (vs tumours) overexpressed EGFR protein. In the paired cell lines, PRC3 and WT8, EGFR protein was elevated post-transcriptionally in the VHL mutant and EGF-stimulated phosphorylation was prolonged. We propose that combined EGFR and mTOR inhibitors may be useful in the subset of RCCs with wt-VHL. However, apparent differences between primary tumours and cell lines require further investigation.

Show MeSH
Related in: MedlinePlus