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Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumours sensitive and resistant to anti-EGFR drugs.

Bianco R, Garofalo S, Rosa R, Damiano V, Gelardi T, Daniele G, Marciano R, Ciardiello F, Tortora G - Br. J. Cancer (2008)

Bottom Line: Everolimus reduces the expression of EGFR-related signalling effectors and VEGF production, inhibiting proliferation and capillary tube formation of endothelial cells, both alone and in combination with gefitinib.Finally, combination of everolimus and gefitinib inhibits growth of GEO and GEO-GR (gefitinib resistant) colon cancer xenografts, activation of signalling proteins and VEGF secretion.Targeting mTOR pathway with everolimus overcomes resistance to EGFR inhibitors and produces a cooperative effect with EGFR inhibitors, providing a valid therapeutic strategy to be tested in a clinical setting.

View Article: PubMed Central - PubMed

Affiliation: Cattedra di Oncologia Medica, Dipartimento Endocrinologia e Oncologia Molecolare e Clinica, Università di Napoli Federico II, via S. Pansini 5, Napoli 80131, Italy.

ABSTRACT
Inhibition of a single transduction pathway is often inefficient due to activation of alternative signalling. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation, survival and angiogenic pathways and has been implicated in the resistance to EGFR inhibitors. Thus, mTOR blockade is pursued to interfere at multiple levels with tumour growth. We used everolimus (RAD001) to inhibit mTOR, alone or in combination with anti-EGFR drugs gefitinib or cetuximab, on human cancer cell lines sensitive and resistant to EGFR inhibitors, both in vitro and in vivo. We demonstrated that everolimus is active against EGFR-resistant cancer cell lines and partially restores the ability of EGFR inhibitors to inhibit growth and survival. Everolimus reduces the expression of EGFR-related signalling effectors and VEGF production, inhibiting proliferation and capillary tube formation of endothelial cells, both alone and in combination with gefitinib. Finally, combination of everolimus and gefitinib inhibits growth of GEO and GEO-GR (gefitinib resistant) colon cancer xenografts, activation of signalling proteins and VEGF secretion. Targeting mTOR pathway with everolimus overcomes resistance to EGFR inhibitors and produces a cooperative effect with EGFR inhibitors, providing a valid therapeutic strategy to be tested in a clinical setting.

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(A) GEO and GEO-GR cells were treated with everolimus (0.1 μM) or gefitinib (5 μM) alone or in combination. The results are statistically significant for everolimus plus gefitinib vs control, vs everolimus alone and vs gefitinib alone (two-sided P<0.0001). For GEO cells, the results are statistically significant for both gefitinib and everolimus vs control (two-sided P<0.0001), whereas for GEO-GR cells only for everolimus vs control (two-sided P<0.0001). (B) Western blotting on total cell lysates from GEO and GEO-GR cells treated with 1 μM gefitinib and/or 0.1 μM everolimus. Lane 1: untreated control; lane 2: gefitinib; lane 3: everolimus; lane 4: gefitinib plus everolimus.
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fig2: (A) GEO and GEO-GR cells were treated with everolimus (0.1 μM) or gefitinib (5 μM) alone or in combination. The results are statistically significant for everolimus plus gefitinib vs control, vs everolimus alone and vs gefitinib alone (two-sided P<0.0001). For GEO cells, the results are statistically significant for both gefitinib and everolimus vs control (two-sided P<0.0001), whereas for GEO-GR cells only for everolimus vs control (two-sided P<0.0001). (B) Western blotting on total cell lysates from GEO and GEO-GR cells treated with 1 μM gefitinib and/or 0.1 μM everolimus. Lane 1: untreated control; lane 2: gefitinib; lane 3: everolimus; lane 4: gefitinib plus everolimus.

Mentions: To verify the effects of everolimus alone or in combination with gefitinib on wild-type GEO and GEO-GR cell lines, we performed a cell-survival assay. As expected, gefitinib (5 μM) induced a 50% cell-survival inhibition in GEO cells, whereas it was totally ineffective on GEO-GR cells. Everolimus (0.1 μM) was effective on GEO cells, with a cell-survival inhibition of about 25%, and it showed a slight activity on GEO-GR cells. Combination of everolimus and gefitinib improved the cell-survival inhibition caused by gefitinib alone in sensitive GEO cells, and it partially reverted the resistance to gefitinib in GEO-GR cells with a cell-survival inhibition of about 30% (Figure 2A). These results refer to the simultaneous administration of the two agents. This modality was chosen because previously we performed a series of experiments to test different sequences of administration of the two drugs, comparing either the use of everolimus 1, 6 or 24 h before gefitinib, or gefitinib before everolimus or their simultaneous treatment. We observed no significant differences among the different modalities used (data not shown).


Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumours sensitive and resistant to anti-EGFR drugs.

Bianco R, Garofalo S, Rosa R, Damiano V, Gelardi T, Daniele G, Marciano R, Ciardiello F, Tortora G - Br. J. Cancer (2008)

(A) GEO and GEO-GR cells were treated with everolimus (0.1 μM) or gefitinib (5 μM) alone or in combination. The results are statistically significant for everolimus plus gefitinib vs control, vs everolimus alone and vs gefitinib alone (two-sided P<0.0001). For GEO cells, the results are statistically significant for both gefitinib and everolimus vs control (two-sided P<0.0001), whereas for GEO-GR cells only for everolimus vs control (two-sided P<0.0001). (B) Western blotting on total cell lysates from GEO and GEO-GR cells treated with 1 μM gefitinib and/or 0.1 μM everolimus. Lane 1: untreated control; lane 2: gefitinib; lane 3: everolimus; lane 4: gefitinib plus everolimus.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2266842&req=5

fig2: (A) GEO and GEO-GR cells were treated with everolimus (0.1 μM) or gefitinib (5 μM) alone or in combination. The results are statistically significant for everolimus plus gefitinib vs control, vs everolimus alone and vs gefitinib alone (two-sided P<0.0001). For GEO cells, the results are statistically significant for both gefitinib and everolimus vs control (two-sided P<0.0001), whereas for GEO-GR cells only for everolimus vs control (two-sided P<0.0001). (B) Western blotting on total cell lysates from GEO and GEO-GR cells treated with 1 μM gefitinib and/or 0.1 μM everolimus. Lane 1: untreated control; lane 2: gefitinib; lane 3: everolimus; lane 4: gefitinib plus everolimus.
Mentions: To verify the effects of everolimus alone or in combination with gefitinib on wild-type GEO and GEO-GR cell lines, we performed a cell-survival assay. As expected, gefitinib (5 μM) induced a 50% cell-survival inhibition in GEO cells, whereas it was totally ineffective on GEO-GR cells. Everolimus (0.1 μM) was effective on GEO cells, with a cell-survival inhibition of about 25%, and it showed a slight activity on GEO-GR cells. Combination of everolimus and gefitinib improved the cell-survival inhibition caused by gefitinib alone in sensitive GEO cells, and it partially reverted the resistance to gefitinib in GEO-GR cells with a cell-survival inhibition of about 30% (Figure 2A). These results refer to the simultaneous administration of the two agents. This modality was chosen because previously we performed a series of experiments to test different sequences of administration of the two drugs, comparing either the use of everolimus 1, 6 or 24 h before gefitinib, or gefitinib before everolimus or their simultaneous treatment. We observed no significant differences among the different modalities used (data not shown).

Bottom Line: Everolimus reduces the expression of EGFR-related signalling effectors and VEGF production, inhibiting proliferation and capillary tube formation of endothelial cells, both alone and in combination with gefitinib.Finally, combination of everolimus and gefitinib inhibits growth of GEO and GEO-GR (gefitinib resistant) colon cancer xenografts, activation of signalling proteins and VEGF secretion.Targeting mTOR pathway with everolimus overcomes resistance to EGFR inhibitors and produces a cooperative effect with EGFR inhibitors, providing a valid therapeutic strategy to be tested in a clinical setting.

View Article: PubMed Central - PubMed

Affiliation: Cattedra di Oncologia Medica, Dipartimento Endocrinologia e Oncologia Molecolare e Clinica, Università di Napoli Federico II, via S. Pansini 5, Napoli 80131, Italy.

ABSTRACT
Inhibition of a single transduction pathway is often inefficient due to activation of alternative signalling. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation, survival and angiogenic pathways and has been implicated in the resistance to EGFR inhibitors. Thus, mTOR blockade is pursued to interfere at multiple levels with tumour growth. We used everolimus (RAD001) to inhibit mTOR, alone or in combination with anti-EGFR drugs gefitinib or cetuximab, on human cancer cell lines sensitive and resistant to EGFR inhibitors, both in vitro and in vivo. We demonstrated that everolimus is active against EGFR-resistant cancer cell lines and partially restores the ability of EGFR inhibitors to inhibit growth and survival. Everolimus reduces the expression of EGFR-related signalling effectors and VEGF production, inhibiting proliferation and capillary tube formation of endothelial cells, both alone and in combination with gefitinib. Finally, combination of everolimus and gefitinib inhibits growth of GEO and GEO-GR (gefitinib resistant) colon cancer xenografts, activation of signalling proteins and VEGF secretion. Targeting mTOR pathway with everolimus overcomes resistance to EGFR inhibitors and produces a cooperative effect with EGFR inhibitors, providing a valid therapeutic strategy to be tested in a clinical setting.

Show MeSH
Related in: MedlinePlus