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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

Regulation of ALK kinase activity by intracellular phosphatases.RH30, RH4 and SH-SY-5Y cells were treated with Pervanadate (A) or agonist mAb46 monoclonal antibody (B). Immunoblottings of total and phosphorylated ALK, c-Met (MET), ERK and AKT proteins are shown. (C) Regulation of inducible ALK phosphorylation and activity. RH30, RH4 and SH-SY-5Y cells were treated with both mAb46 (3 μg/ml) and Pervanadate (50 μM) to measure ALK phosphorylation and signalling. To inhibit ALK, the cells were pre-incubated with antagonist mAb30 antibody (2 μg/ml) or with Crizotinib inhibitor (1 μM). Protein lysates were subjected to Western blot analysis using antibodies specific for total and phosphorylated ALK and ERK proteins, as described.
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pone.0132330.g003: Regulation of ALK kinase activity by intracellular phosphatases.RH30, RH4 and SH-SY-5Y cells were treated with Pervanadate (A) or agonist mAb46 monoclonal antibody (B). Immunoblottings of total and phosphorylated ALK, c-Met (MET), ERK and AKT proteins are shown. (C) Regulation of inducible ALK phosphorylation and activity. RH30, RH4 and SH-SY-5Y cells were treated with both mAb46 (3 μg/ml) and Pervanadate (50 μM) to measure ALK phosphorylation and signalling. To inhibit ALK, the cells were pre-incubated with antagonist mAb30 antibody (2 μg/ml) or with Crizotinib inhibitor (1 μM). Protein lysates were subjected to Western blot analysis using antibodies specific for total and phosphorylated ALK and ERK proteins, as described.

Mentions: The generation of intracellular signalling depends on balanced activities of constitutively active RTKs and protein tyrosine phosphatases (PTPases) [50]. As such, basal activity of weakly expressed RTKs may be not discernable due to permanent dephosphorylation by intracellular PTPases, whereas at high expression levels detection of receptor phosphorylation is feasible [46,47,51]. Thus, to investigate the impact of phosphatases on ALK phosphorylation and activity, pervanadate inhibitor was administered to the cells and the effect compared with that observed after exposure to mAb46. We demonstrated that PTPase inhibition clearly increased ALK phosphorylation in RH30 and SH-SY5Y cells, and led to activation of ERK and AKT target proteins (Fig 3A). However, pharmacological inhibition of intracellular phosphatases by pervanadate promoted a general RTKs activation in these cells, since stimulation of c-Met receptor, as well as phosphorylation of downstream ERK kinase, was observed independently of ALK expression and activity (Fig 3A, RH30 vs. RH4). In contrast, mAb46 treatment selectively activated ALK and had no influence on the activity and signal processing of other RTKs (Fig 3B). Indeed, when ALK antagonist mAb30 antibody or tyrosine kinase inhibitor crizotinib were administered prior to mAb46 or pervanadate exposure, activation of ALK was completely prevented, whereas downregulation of ALK-dependent phosphorylation of ERK was observed only in mAb46-treated cells (Fig 3C).


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)

Regulation of ALK kinase activity by intracellular phosphatases.RH30, RH4 and SH-SY-5Y cells were treated with Pervanadate (A) or agonist mAb46 monoclonal antibody (B). Immunoblottings of total and phosphorylated ALK, c-Met (MET), ERK and AKT proteins are shown. (C) Regulation of inducible ALK phosphorylation and activity. RH30, RH4 and SH-SY-5Y cells were treated with both mAb46 (3 μg/ml) and Pervanadate (50 μM) to measure ALK phosphorylation and signalling. To inhibit ALK, the cells were pre-incubated with antagonist mAb30 antibody (2 μg/ml) or with Crizotinib inhibitor (1 μM). Protein lysates were subjected to Western blot analysis using antibodies specific for total and phosphorylated ALK and ERK proteins, as described.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132330.g003: Regulation of ALK kinase activity by intracellular phosphatases.RH30, RH4 and SH-SY-5Y cells were treated with Pervanadate (A) or agonist mAb46 monoclonal antibody (B). Immunoblottings of total and phosphorylated ALK, c-Met (MET), ERK and AKT proteins are shown. (C) Regulation of inducible ALK phosphorylation and activity. RH30, RH4 and SH-SY-5Y cells were treated with both mAb46 (3 μg/ml) and Pervanadate (50 μM) to measure ALK phosphorylation and signalling. To inhibit ALK, the cells were pre-incubated with antagonist mAb30 antibody (2 μg/ml) or with Crizotinib inhibitor (1 μM). Protein lysates were subjected to Western blot analysis using antibodies specific for total and phosphorylated ALK and ERK proteins, as described.
Mentions: The generation of intracellular signalling depends on balanced activities of constitutively active RTKs and protein tyrosine phosphatases (PTPases) [50]. As such, basal activity of weakly expressed RTKs may be not discernable due to permanent dephosphorylation by intracellular PTPases, whereas at high expression levels detection of receptor phosphorylation is feasible [46,47,51]. Thus, to investigate the impact of phosphatases on ALK phosphorylation and activity, pervanadate inhibitor was administered to the cells and the effect compared with that observed after exposure to mAb46. We demonstrated that PTPase inhibition clearly increased ALK phosphorylation in RH30 and SH-SY5Y cells, and led to activation of ERK and AKT target proteins (Fig 3A). However, pharmacological inhibition of intracellular phosphatases by pervanadate promoted a general RTKs activation in these cells, since stimulation of c-Met receptor, as well as phosphorylation of downstream ERK kinase, was observed independently of ALK expression and activity (Fig 3A, RH30 vs. RH4). In contrast, mAb46 treatment selectively activated ALK and had no influence on the activity and signal processing of other RTKs (Fig 3B). Indeed, when ALK antagonist mAb30 antibody or tyrosine kinase inhibitor crizotinib were administered prior to mAb46 or pervanadate exposure, activation of ALK was completely prevented, whereas downregulation of ALK-dependent phosphorylation of ERK was observed only in mAb46-treated cells (Fig 3C).

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