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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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Related in: MedlinePlus

ELISA assays for hVEGF on conditioned media from (A) cancer cells treated with everolimus (0.1 μM) and from (B) GEO and (C) GEO-GR cancer cells treated with everolimus (0.1 μM) and gefitinib (5 μM) alone or in combination. HUVEC cells were treated with everolimus from 1 nM to 1 μM (D) or with everolimus 0.01 μM and gefitinib 1 μM alone or the combination (E). HUVECs were incubated on solidified matrigel in presence of everolimus 0.1 μM, gefitinib 5 μM or the combination. The positive control was matrigel with VEGF 100 ng ml−1. Photographies were performed at 0 and 24 h (F). All the results are statistically significant for each treatment vs control (two-sided P<0.0001).
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fig3: ELISA assays for hVEGF on conditioned media from (A) cancer cells treated with everolimus (0.1 μM) and from (B) GEO and (C) GEO-GR cancer cells treated with everolimus (0.1 μM) and gefitinib (5 μM) alone or in combination. HUVEC cells were treated with everolimus from 1 nM to 1 μM (D) or with everolimus 0.01 μM and gefitinib 1 μM alone or the combination (E). HUVECs were incubated on solidified matrigel in presence of everolimus 0.1 μM, gefitinib 5 μM or the combination. The positive control was matrigel with VEGF 100 ng ml−1. Photographies were performed at 0 and 24 h (F). All the results are statistically significant for each treatment vs control (two-sided P<0.0001).

Mentions: Based on the reported ability of mTOR to affect VEGF levels (Guba et al, 2002), we evaluated the effect of everolimus on VEGF production in our cancer cell lines. As shown in Figure 3A, everolimus reduces human VEGF (hVEGF) secretion of 20–45% both in wild type and in resistant cell lines, compared with untreated controls. We then measured hVEGF by ELISA assay on conditioned media derived from GEO and GEO-GR cancer cell culture treated for 24 h with everolimus (0.1 μM) and gefitinib (5 μM) alone or in combination. Gefitinib significantly reduces hVEGF levels only in wild-type GEO cells, whereas everolimus is active in both cell lines, especially in resistant GEO-GR cells. Combined treatment with gefitinib and everolimus causes a strong reduction of hVEGF levels both in GEO and in GEO-GR cells (Figure 3B and C).


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)

ELISA assays for hVEGF on conditioned media from (A) cancer cells treated with everolimus (0.1 μM) and from (B) GEO and (C) GEO-GR cancer cells treated with everolimus (0.1 μM) and gefitinib (5 μM) alone or in combination. HUVEC cells were treated with everolimus from 1 nM to 1 μM (D) or with everolimus 0.01 μM and gefitinib 1 μM alone or the combination (E). HUVECs were incubated on solidified matrigel in presence of everolimus 0.1 μM, gefitinib 5 μM or the combination. The positive control was matrigel with VEGF 100 ng ml−1. Photographies were performed at 0 and 24 h (F). All the results are statistically significant for each treatment vs control (two-sided P<0.0001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266842&req=5

fig3: ELISA assays for hVEGF on conditioned media from (A) cancer cells treated with everolimus (0.1 μM) and from (B) GEO and (C) GEO-GR cancer cells treated with everolimus (0.1 μM) and gefitinib (5 μM) alone or in combination. HUVEC cells were treated with everolimus from 1 nM to 1 μM (D) or with everolimus 0.01 μM and gefitinib 1 μM alone or the combination (E). HUVECs were incubated on solidified matrigel in presence of everolimus 0.1 μM, gefitinib 5 μM or the combination. The positive control was matrigel with VEGF 100 ng ml−1. Photographies were performed at 0 and 24 h (F). All the results are statistically significant for each treatment vs control (two-sided P<0.0001).
Mentions: Based on the reported ability of mTOR to affect VEGF levels (Guba et al, 2002), we evaluated the effect of everolimus on VEGF production in our cancer cell lines. As shown in Figure 3A, everolimus reduces human VEGF (hVEGF) secretion of 20–45% both in wild type and in resistant cell lines, compared with untreated controls. We then measured hVEGF by ELISA assay on conditioned media derived from GEO and GEO-GR cancer cell culture treated for 24 h with everolimus (0.1 μM) and gefitinib (5 μM) alone or in combination. Gefitinib significantly reduces hVEGF levels only in wild-type GEO cells, whereas everolimus is active in both cell lines, especially in resistant GEO-GR cells. Combined treatment with gefitinib and everolimus causes a strong reduction of hVEGF levels both in GEO and in GEO-GR cells (Figure 3B and C).

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