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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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Polysome analysis of WT8 cells. WT8 cells were grown to 70% confluency, then treated for 2 h with DMSO, rapamycin (10 nM), Iressa (10 μM) or both, and harvested for polysome fractionation. A representative A280 fractionation profile is shown along with the fractions pooled and the results from quantitative RT–PCR analysis.
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fig5: Polysome analysis of WT8 cells. WT8 cells were grown to 70% confluency, then treated for 2 h with DMSO, rapamycin (10 nM), Iressa (10 μM) or both, and harvested for polysome fractionation. A representative A280 fractionation profile is shown along with the fractions pooled and the results from quantitative RT–PCR analysis.

Mentions: To determine whether the elevated EGFR protein in PRC3 was the result of mRNA differences, possibly due to clonal variation, we examined message levels by real-time RT–PCR in parental 786-O, PRC3 and WT8 cells. The analyses, performed in triplicate, demonstrated that there were no differences among the cell lines (Table 3). Like the original series of RCCs examined, ErbB-2, ErbB-3 and ErbB-4 mRNA levels were barely detectable and corresponding proteins were undetectable by Western analysis. In contrast, VEGF expression, as expected, was higher in the VHL mutants 786-O and PRC3 than WT8. These results indicate that the observed differences in EGFR protein levels are post-transcriptional, possibly involving protein translation initiation or degradation. To examine the question of protein translation initiation, we isolated polysome fractions from WT8 and PRC3 cells and analysed these by quantitative real-time RT–PCR (Figure 5). No reproducible differences were identified in ribosome-bound EGFR mRNA between these cell lines. Furthermore, short-term treatment with rapamycin or Iressa had no consistent effects on polysome-associated EGFR message (not shown). These results suggest that the difference in EGFR protein levels (and phosphorylation duration) between PRC3 and WT8 involves internalisation or degradation.


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)

Polysome analysis of WT8 cells. WT8 cells were grown to 70% confluency, then treated for 2 h with DMSO, rapamycin (10 nM), Iressa (10 μM) or both, and harvested for polysome fractionation. A representative A280 fractionation profile is shown along with the fractions pooled and the results from quantitative RT–PCR analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361810&req=5

fig5: Polysome analysis of WT8 cells. WT8 cells were grown to 70% confluency, then treated for 2 h with DMSO, rapamycin (10 nM), Iressa (10 μM) or both, and harvested for polysome fractionation. A representative A280 fractionation profile is shown along with the fractions pooled and the results from quantitative RT–PCR analysis.
Mentions: To determine whether the elevated EGFR protein in PRC3 was the result of mRNA differences, possibly due to clonal variation, we examined message levels by real-time RT–PCR in parental 786-O, PRC3 and WT8 cells. The analyses, performed in triplicate, demonstrated that there were no differences among the cell lines (Table 3). Like the original series of RCCs examined, ErbB-2, ErbB-3 and ErbB-4 mRNA levels were barely detectable and corresponding proteins were undetectable by Western analysis. In contrast, VEGF expression, as expected, was higher in the VHL mutants 786-O and PRC3 than WT8. These results indicate that the observed differences in EGFR protein levels are post-transcriptional, possibly involving protein translation initiation or degradation. To examine the question of protein translation initiation, we isolated polysome fractions from WT8 and PRC3 cells and analysed these by quantitative real-time RT–PCR (Figure 5). No reproducible differences were identified in ribosome-bound EGFR mRNA between these cell lines. Furthermore, short-term treatment with rapamycin or Iressa had no consistent effects on polysome-associated EGFR message (not shown). These results suggest that the difference in EGFR protein levels (and phosphorylation duration) between PRC3 and WT8 involves internalisation or degradation.

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