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Vertical suppression of the EGFR pathway prevents onset of resistance in colorectal cancers.

Misale S, Bozic I, Tong J, Peraza-Penton A, Lallo A, Baldi F, Lin KH, Truini M, Trusolino L, Bertotti A, Di Nicolantonio F, Nowak MA, Zhang L, Wood KC, Bardelli A - Nat Commun (2015)

Bottom Line: Molecular targeted drugs are clinically effective anti-cancer therapies.Following treatment of CRC cells with anti-EGFR, anti-MEK or the combination of the two drugs, we find that EGFR blockade alone triggers acquired resistance in weeks, while combinatorial treatment does not induce resistance.Mechanistically, we find that the EGFR-MEK Combo blockade triggers Bcl-2 and Mcl-1 downregulation and initiates apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Candiolo Cancer Institute - Fondazione Piemontese per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Candiolo, Torino, 10060, Italy.

ABSTRACT
Molecular targeted drugs are clinically effective anti-cancer therapies. However, tumours treated with single agents usually develop resistance. Here we use colorectal cancer (CRC) as a model to study how the acquisition of resistance to EGFR-targeted therapies can be restrained. Pathway-oriented genetic screens reveal that CRC cells escape from EGFR blockade by downstream activation of RAS-MEK signalling. Following treatment of CRC cells with anti-EGFR, anti-MEK or the combination of the two drugs, we find that EGFR blockade alone triggers acquired resistance in weeks, while combinatorial treatment does not induce resistance. In patient-derived xenografts, EGFR-MEK combination prevents the development of resistance. We employ mathematical modelling to provide a quantitative understanding of the dynamics of response and resistance to these single and combination therapies. Mechanistically, we find that the EGFR-MEK Combo blockade triggers Bcl-2 and Mcl-1 downregulation and initiates apoptosis. These results provide the rationale for clinical trials aimed at preventing rather than intercepting resistance.

No MeSH data available.


Related in: MedlinePlus

Biochemical analysis and apoptosis evaluation of CRC cells treated with EGFR and/or MEK inhibitors.CCK81 (a) and DiFi (b) were treated with cetuximab (Cmab, 340 nM), pimasertib (Pima, 250 nM), or with the combo of the two drugs at the indicated time points, whole-cell extracts were subjected to western blot analysis and compared with untreated cells with phospho-EGFR (Tyr 1068), total EGFR, total AKT and phospho-AKT (Ser 473), total ERK1/2 and phospho-ERK1/2 antibodies. Actin was included as a loading control. (c,d) Concomitant blockade of EGFR and MEK triggers apoptosis. CCK81 and DiFi cells were treated at different time points with cetuximab, pimasertib or both. Nuclei fragmentation (c) and caspase-3 activation (d) were measured. Rates of apoptosis due to combination are approximatively twofold higher compared with those due to cetuximab, in line with our model predictions. (e) Apoptotic pathway activation. The indicated CRC cell lines were treated with cetuximab (Cmab, 340 nM), pimasertib (Pima, 250 nM), or with the two drugs for 48 h. Whole-cell extracts were subjected to Western blot analysis and compared with untreated cells using BAK, Bax, Bid, NOXA, PUMA, Bim, Bcl-2, Mcl-1, Bcl-XL and active caspase-3 antibodies. Actin was included as a loading control.
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f7: Biochemical analysis and apoptosis evaluation of CRC cells treated with EGFR and/or MEK inhibitors.CCK81 (a) and DiFi (b) were treated with cetuximab (Cmab, 340 nM), pimasertib (Pima, 250 nM), or with the combo of the two drugs at the indicated time points, whole-cell extracts were subjected to western blot analysis and compared with untreated cells with phospho-EGFR (Tyr 1068), total EGFR, total AKT and phospho-AKT (Ser 473), total ERK1/2 and phospho-ERK1/2 antibodies. Actin was included as a loading control. (c,d) Concomitant blockade of EGFR and MEK triggers apoptosis. CCK81 and DiFi cells were treated at different time points with cetuximab, pimasertib or both. Nuclei fragmentation (c) and caspase-3 activation (d) were measured. Rates of apoptosis due to combination are approximatively twofold higher compared with those due to cetuximab, in line with our model predictions. (e) Apoptotic pathway activation. The indicated CRC cell lines were treated with cetuximab (Cmab, 340 nM), pimasertib (Pima, 250 nM), or with the two drugs for 48 h. Whole-cell extracts were subjected to Western blot analysis and compared with untreated cells using BAK, Bax, Bid, NOXA, PUMA, Bim, Bcl-2, Mcl-1, Bcl-XL and active caspase-3 antibodies. Actin was included as a loading control.

Mentions: Experiments in cells and tumourgrafts and mathematical models indicate that combinatorial blockade of EGFR and MEK impairs the emergence of resistant clones in vitro and in vivo. At first this tactic may seem counter-intuitive. Why should it be necessary to intercept two nodes in the same pathway? To shed light on the remarkable efficacy of EGFR-MEK concomitant inhibition we performed biochemical analyses. The experiments of Fig. 2 indicate that CRC cells sensitive to EGFR blockade are virtually unaffected by MEK inhibition and quickly resume exponential growth in the presence of MEK suppression. Biochemical profiling of EGFR downstream pathways in DiFi and CCK81 cells revealed that MEK inhibition induces EGFR and AKT activation (p-Y1068 and p-S473) and is unable to switch off ERK phosphorylation, thus suggesting an adaptive escape from MEK blockade (Fig. 7a,b and Supplementary Fig 7a,b).


Vertical suppression of the EGFR pathway prevents onset of resistance in colorectal cancers.

Misale S, Bozic I, Tong J, Peraza-Penton A, Lallo A, Baldi F, Lin KH, Truini M, Trusolino L, Bertotti A, Di Nicolantonio F, Nowak MA, Zhang L, Wood KC, Bardelli A - Nat Commun (2015)

Biochemical analysis and apoptosis evaluation of CRC cells treated with EGFR and/or MEK inhibitors.CCK81 (a) and DiFi (b) were treated with cetuximab (Cmab, 340 nM), pimasertib (Pima, 250 nM), or with the combo of the two drugs at the indicated time points, whole-cell extracts were subjected to western blot analysis and compared with untreated cells with phospho-EGFR (Tyr 1068), total EGFR, total AKT and phospho-AKT (Ser 473), total ERK1/2 and phospho-ERK1/2 antibodies. Actin was included as a loading control. (c,d) Concomitant blockade of EGFR and MEK triggers apoptosis. CCK81 and DiFi cells were treated at different time points with cetuximab, pimasertib or both. Nuclei fragmentation (c) and caspase-3 activation (d) were measured. Rates of apoptosis due to combination are approximatively twofold higher compared with those due to cetuximab, in line with our model predictions. (e) Apoptotic pathway activation. The indicated CRC cell lines were treated with cetuximab (Cmab, 340 nM), pimasertib (Pima, 250 nM), or with the two drugs for 48 h. Whole-cell extracts were subjected to Western blot analysis and compared with untreated cells using BAK, Bax, Bid, NOXA, PUMA, Bim, Bcl-2, Mcl-1, Bcl-XL and active caspase-3 antibodies. Actin was included as a loading control.
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Related In: Results  -  Collection

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f7: Biochemical analysis and apoptosis evaluation of CRC cells treated with EGFR and/or MEK inhibitors.CCK81 (a) and DiFi (b) were treated with cetuximab (Cmab, 340 nM), pimasertib (Pima, 250 nM), or with the combo of the two drugs at the indicated time points, whole-cell extracts were subjected to western blot analysis and compared with untreated cells with phospho-EGFR (Tyr 1068), total EGFR, total AKT and phospho-AKT (Ser 473), total ERK1/2 and phospho-ERK1/2 antibodies. Actin was included as a loading control. (c,d) Concomitant blockade of EGFR and MEK triggers apoptosis. CCK81 and DiFi cells were treated at different time points with cetuximab, pimasertib or both. Nuclei fragmentation (c) and caspase-3 activation (d) were measured. Rates of apoptosis due to combination are approximatively twofold higher compared with those due to cetuximab, in line with our model predictions. (e) Apoptotic pathway activation. The indicated CRC cell lines were treated with cetuximab (Cmab, 340 nM), pimasertib (Pima, 250 nM), or with the two drugs for 48 h. Whole-cell extracts were subjected to Western blot analysis and compared with untreated cells using BAK, Bax, Bid, NOXA, PUMA, Bim, Bcl-2, Mcl-1, Bcl-XL and active caspase-3 antibodies. Actin was included as a loading control.
Mentions: Experiments in cells and tumourgrafts and mathematical models indicate that combinatorial blockade of EGFR and MEK impairs the emergence of resistant clones in vitro and in vivo. At first this tactic may seem counter-intuitive. Why should it be necessary to intercept two nodes in the same pathway? To shed light on the remarkable efficacy of EGFR-MEK concomitant inhibition we performed biochemical analyses. The experiments of Fig. 2 indicate that CRC cells sensitive to EGFR blockade are virtually unaffected by MEK inhibition and quickly resume exponential growth in the presence of MEK suppression. Biochemical profiling of EGFR downstream pathways in DiFi and CCK81 cells revealed that MEK inhibition induces EGFR and AKT activation (p-Y1068 and p-S473) and is unable to switch off ERK phosphorylation, thus suggesting an adaptive escape from MEK blockade (Fig. 7a,b and Supplementary Fig 7a,b).

Bottom Line: Molecular targeted drugs are clinically effective anti-cancer therapies.Following treatment of CRC cells with anti-EGFR, anti-MEK or the combination of the two drugs, we find that EGFR blockade alone triggers acquired resistance in weeks, while combinatorial treatment does not induce resistance.Mechanistically, we find that the EGFR-MEK Combo blockade triggers Bcl-2 and Mcl-1 downregulation and initiates apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Candiolo Cancer Institute - Fondazione Piemontese per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Candiolo, Torino, 10060, Italy.

ABSTRACT
Molecular targeted drugs are clinically effective anti-cancer therapies. However, tumours treated with single agents usually develop resistance. Here we use colorectal cancer (CRC) as a model to study how the acquisition of resistance to EGFR-targeted therapies can be restrained. Pathway-oriented genetic screens reveal that CRC cells escape from EGFR blockade by downstream activation of RAS-MEK signalling. Following treatment of CRC cells with anti-EGFR, anti-MEK or the combination of the two drugs, we find that EGFR blockade alone triggers acquired resistance in weeks, while combinatorial treatment does not induce resistance. In patient-derived xenografts, EGFR-MEK combination prevents the development of resistance. We employ mathematical modelling to provide a quantitative understanding of the dynamics of response and resistance to these single and combination therapies. Mechanistically, we find that the EGFR-MEK Combo blockade triggers Bcl-2 and Mcl-1 downregulation and initiates apoptosis. These results provide the rationale for clinical trials aimed at preventing rather than intercepting resistance.

No MeSH data available.


Related in: MedlinePlus