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Understanding the Interplay between Expression, Mutation and Activity of ALK Receptor in Rhabdomyosarcoma Cells for Clinical Application of Small-Molecule Inhibitors.

Peron M, Lovisa F, Poli E, Basso G, Bonvini P - PLoS ONE (2015)

Bottom Line: Receptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation.We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity.However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity.

View Article: PubMed Central - PubMed

Affiliation: Clinica di Oncoematologia Pediatrica di Padova, Azienda Ospedaliera-Università di Padova, Padua, Italy.

ABSTRACT

Background: Receptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation. This concept is essential from a therapeutic standpoint, as clinical evidence indicates that tumours carrying deregulated RTKs are particularly susceptible to their activity but also to their inhibition. Rhabdomyosarcoma (RMS) is an aggressive childhood cancer where emerging therapies rely on the use kinase inhibitors, and among druggable kinases ALK represents a potential therapeutic target to commit efforts against. However, the functional relevance of ALK in RMS is not known, likewise the multi-component deregulated RTK profile to which ALK belongs.

Methods: In this study we used RMS cell lines representative of the alveolar and embrional histotype and looked at ALK intracellular localization, activity and cell signalling.

Results: We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity. Indeed, increase of ALK phosphorylation was observed upon PTPase inhibition, as well as after ligand binding or protein overexpression. In these conditions, ALK signalling proceeded through the MAPK/ERK and PI3K/AKT pathways, and it was susceptible to ATP-competitive inhibitors exposure. However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity. Indeed, analysis of baseline and inducible RTK phosphorylation confirmed that RMS cells were susceptible to ALK kinase inhibitors even in the absence of the primary intended target, due to the presence of compensatory RTKs signalling pathways.

Conclusions: These data, hence, provided evidences of a potentially active role of ALK in RMS cells, but also suggest caution in considering ALK a major therapeutic target in this malignancy, particularly if expression and activity cannot be accurately determined.

No MeSH data available.


Related in: MedlinePlus

Multitarget inhibitory activity of crizotinib and TAE684 in RMS cells.(A) Western blot analysis of c-Met, IGF-1R and EGFR ligand-dependent phosphorylation. RMS cells were exposed to cognate receptor ligands (50 ng/ml HGF; 200 ng/ml IGF-I; 50 ng/ml EGF), in the presence and absence of Crizotinib or TAE684 inhibitors. Lysates were probed with antibodies directed against the specified proteins and γ-Tubulin. (B) Western blot analysis of c-Met, IGF-1R and EGFR ligand-dependent signalling. Effects on ERK and AKT phosphorylation were assessed using the specified antibodies. γ-Tubulin was used as loading control. (C) Growth inhibition of RH30 and RD cells after 24H-drug treatment, in the presence or absence of HGF, IGF-I or EGF growth factors. Growth curves show average values and IC50 values of triplicates from one representative experiment out of two, whereas bar graphs aside represent combined exposure to HGF, IGF-1 and EGF growth factors in the presence or absence of 5 μM Crizotinib, TAE684 or both for 24 hours.
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pone.0132330.g006: Multitarget inhibitory activity of crizotinib and TAE684 in RMS cells.(A) Western blot analysis of c-Met, IGF-1R and EGFR ligand-dependent phosphorylation. RMS cells were exposed to cognate receptor ligands (50 ng/ml HGF; 200 ng/ml IGF-I; 50 ng/ml EGF), in the presence and absence of Crizotinib or TAE684 inhibitors. Lysates were probed with antibodies directed against the specified proteins and γ-Tubulin. (B) Western blot analysis of c-Met, IGF-1R and EGFR ligand-dependent signalling. Effects on ERK and AKT phosphorylation were assessed using the specified antibodies. γ-Tubulin was used as loading control. (C) Growth inhibition of RH30 and RD cells after 24H-drug treatment, in the presence or absence of HGF, IGF-I or EGF growth factors. Growth curves show average values and IC50 values of triplicates from one representative experiment out of two, whereas bar graphs aside represent combined exposure to HGF, IGF-1 and EGF growth factors in the presence or absence of 5 μM Crizotinib, TAE684 or both for 24 hours.

Mentions: Cancer cells typically express multiple RTKs that mediate signals through common downstream survival factors, and can be rescued from drug sensitivity by simply exposing them to one or more receptor ligands [59,60,61]. Therefore, to investigate the possibility that potency of ALK inhibitors in RMS cells might be the result of inhibition of other targets, c-MET, IGF-1R and EGFR RTKs were stimulated by cognate ligands and drug-induced perturbation of cell signaling was assessed. Similar to ALK, basal phosphorylation of these receptors was variable in the three RMS cell lines, and in some cases too weak to be discernable without growth factor stimulation. Analysis of c-Met, IGF-1R and EGFR phosphorylation, however, confirmed their activation by cognate ligands (HGF, IGF-I and EGF, respectively) and their expected sensitivity to ALK inhibitors. In particular, HGF stimulated c-Met receptor in the presence of TAE684 but not of crizotinib, IGF-I-dependent activation of IGF-1R was sensitive to both drugs, whereas activated EGFR was resistant either to Crizotinib or TAE684 (Fig 6A). With respect to cell signaling, HGF administration activated ERK (pERK) in all 3 cell lines, whereas EGF stimulated ERK phosphorylation mainly in RD cells (Fig 6B). IGF-I, in contrast, failed to generate additional signaling output despite IGF-1R receptor activation. Moreover, consistent with kinase inhibitors specificity, HGF activated ERK in the presence of TAE684 only (Fig 6B, lanes 2–3 and 9–10), EGF induced ERK phosphorylation in the presence of both inhibitors (Fig 6B, lanes 6–7 and 13–14), whereas IGF was unable to sustain cell signaling during drug treatment (Fig 6B, lanes 4–5 and 11–12). None of these factors, however, were able to completely rescue RMS cells from signaling downregulation, as shown by the almost complete inhibition of AKT phosphorylation in these conditions. Ligand exposure, in fact, had a limited effect on growth and survival of RMS cells, since, except in RD cells exposed to EGF, growth inhibition progressed independently of receptor activation (Fig 6C), and even increased when drug treatments were combined (Fig 6C, bar graphs).


Understanding the Interplay between Expression, Mutation and Activity of ALK Receptor in Rhabdomyosarcoma Cells for Clinical Application of Small-Molecule Inhibitors.

Peron M, Lovisa F, Poli E, Basso G, Bonvini P - PLoS ONE (2015)

Multitarget inhibitory activity of crizotinib and TAE684 in RMS cells.(A) Western blot analysis of c-Met, IGF-1R and EGFR ligand-dependent phosphorylation. RMS cells were exposed to cognate receptor ligands (50 ng/ml HGF; 200 ng/ml IGF-I; 50 ng/ml EGF), in the presence and absence of Crizotinib or TAE684 inhibitors. Lysates were probed with antibodies directed against the specified proteins and γ-Tubulin. (B) Western blot analysis of c-Met, IGF-1R and EGFR ligand-dependent signalling. Effects on ERK and AKT phosphorylation were assessed using the specified antibodies. γ-Tubulin was used as loading control. (C) Growth inhibition of RH30 and RD cells after 24H-drug treatment, in the presence or absence of HGF, IGF-I or EGF growth factors. Growth curves show average values and IC50 values of triplicates from one representative experiment out of two, whereas bar graphs aside represent combined exposure to HGF, IGF-1 and EGF growth factors in the presence or absence of 5 μM Crizotinib, TAE684 or both for 24 hours.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493009&req=5

pone.0132330.g006: Multitarget inhibitory activity of crizotinib and TAE684 in RMS cells.(A) Western blot analysis of c-Met, IGF-1R and EGFR ligand-dependent phosphorylation. RMS cells were exposed to cognate receptor ligands (50 ng/ml HGF; 200 ng/ml IGF-I; 50 ng/ml EGF), in the presence and absence of Crizotinib or TAE684 inhibitors. Lysates were probed with antibodies directed against the specified proteins and γ-Tubulin. (B) Western blot analysis of c-Met, IGF-1R and EGFR ligand-dependent signalling. Effects on ERK and AKT phosphorylation were assessed using the specified antibodies. γ-Tubulin was used as loading control. (C) Growth inhibition of RH30 and RD cells after 24H-drug treatment, in the presence or absence of HGF, IGF-I or EGF growth factors. Growth curves show average values and IC50 values of triplicates from one representative experiment out of two, whereas bar graphs aside represent combined exposure to HGF, IGF-1 and EGF growth factors in the presence or absence of 5 μM Crizotinib, TAE684 or both for 24 hours.
Mentions: Cancer cells typically express multiple RTKs that mediate signals through common downstream survival factors, and can be rescued from drug sensitivity by simply exposing them to one or more receptor ligands [59,60,61]. Therefore, to investigate the possibility that potency of ALK inhibitors in RMS cells might be the result of inhibition of other targets, c-MET, IGF-1R and EGFR RTKs were stimulated by cognate ligands and drug-induced perturbation of cell signaling was assessed. Similar to ALK, basal phosphorylation of these receptors was variable in the three RMS cell lines, and in some cases too weak to be discernable without growth factor stimulation. Analysis of c-Met, IGF-1R and EGFR phosphorylation, however, confirmed their activation by cognate ligands (HGF, IGF-I and EGF, respectively) and their expected sensitivity to ALK inhibitors. In particular, HGF stimulated c-Met receptor in the presence of TAE684 but not of crizotinib, IGF-I-dependent activation of IGF-1R was sensitive to both drugs, whereas activated EGFR was resistant either to Crizotinib or TAE684 (Fig 6A). With respect to cell signaling, HGF administration activated ERK (pERK) in all 3 cell lines, whereas EGF stimulated ERK phosphorylation mainly in RD cells (Fig 6B). IGF-I, in contrast, failed to generate additional signaling output despite IGF-1R receptor activation. Moreover, consistent with kinase inhibitors specificity, HGF activated ERK in the presence of TAE684 only (Fig 6B, lanes 2–3 and 9–10), EGF induced ERK phosphorylation in the presence of both inhibitors (Fig 6B, lanes 6–7 and 13–14), whereas IGF was unable to sustain cell signaling during drug treatment (Fig 6B, lanes 4–5 and 11–12). None of these factors, however, were able to completely rescue RMS cells from signaling downregulation, as shown by the almost complete inhibition of AKT phosphorylation in these conditions. Ligand exposure, in fact, had a limited effect on growth and survival of RMS cells, since, except in RD cells exposed to EGF, growth inhibition progressed independently of receptor activation (Fig 6C), and even increased when drug treatments were combined (Fig 6C, bar graphs).

Bottom Line: Receptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation.We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity.However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity.

View Article: PubMed Central - PubMed

Affiliation: Clinica di Oncoematologia Pediatrica di Padova, Azienda Ospedaliera-Università di Padova, Padua, Italy.

ABSTRACT

Background: Receptor tyrosine kinases (RTKs) have a central role in cancer initiation and progression, since changes in their expression and activity potentially results in cell transformation. This concept is essential from a therapeutic standpoint, as clinical evidence indicates that tumours carrying deregulated RTKs are particularly susceptible to their activity but also to their inhibition. Rhabdomyosarcoma (RMS) is an aggressive childhood cancer where emerging therapies rely on the use kinase inhibitors, and among druggable kinases ALK represents a potential therapeutic target to commit efforts against. However, the functional relevance of ALK in RMS is not known, likewise the multi-component deregulated RTK profile to which ALK belongs.

Methods: In this study we used RMS cell lines representative of the alveolar and embrional histotype and looked at ALK intracellular localization, activity and cell signalling.

Results: We found that ALK was properly located at the plasma membrane of RMS cells, though in an unphosphorylated and inactive state due to intracellular tyrosine phosphatases (PTPases) activity. Indeed, increase of ALK phosphorylation was observed upon PTPase inhibition, as well as after ligand binding or protein overexpression. In these conditions, ALK signalling proceeded through the MAPK/ERK and PI3K/AKT pathways, and it was susceptible to ATP-competitive inhibitors exposure. However, drug-induced growth inhibition, cell cycle arrest and apoptosis did not correlate with ALK expression only, but relied also on the expression of other RTKs with akin drug binding affinity. Indeed, analysis of baseline and inducible RTK phosphorylation confirmed that RMS cells were susceptible to ALK kinase inhibitors even in the absence of the primary intended target, due to the presence of compensatory RTKs signalling pathways.

Conclusions: These data, hence, provided evidences of a potentially active role of ALK in RMS cells, but also suggest caution in considering ALK a major therapeutic target in this malignancy, particularly if expression and activity cannot be accurately determined.

No MeSH data available.


Related in: MedlinePlus