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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

Kinetics of ALK receptor stimulation and signalling.(A) Antibody-dependent ALK activation was induced by exposing RMS (RH30, RH4) and NB (SH-SY5Y) cells to increasing concentration of mAb46 (0.1–1 μg/ml). Cell lysates were subjected to Western blot analysis using polyclonal antibodies for total and phosphorylated ALK or ERK proteins. Arrowheads indicate membrane-bound (closed) and cytoplasmic (open) full-length ALK kinase, respectively. (B) Time-course analysis of ALK phosphorylation after antibody mAb46 treatment. RH30, SH-SY5Y and RH4 cells were exposed to 1 μg/ml mAb46 for increasing time intervals, and ALK phosphorylation was assessed by Western blot analysis at each time point. Phosphorylation of ERK is also shown. Proteins band density was measured and expressed as fold(s) of control in graphs. Phosphorylated ALK and ERK values were graphed using different scale options. (C) Ligand-independent ALK receptor activity. Relative expression and phosphorylation of ALK RTK was assessed in RH30, SH-SY5Y and RH4 cells after transfection with wild-type (WT) or mutant (F1174L and R1275Q) ALK expression plasmids. Expression and phosphorylation of ALK, ERK, AKT and STAT3 proteins was assessed, using γ-Tubulin as loading control.
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pone.0132330.g002: Kinetics of ALK receptor stimulation and signalling.(A) Antibody-dependent ALK activation was induced by exposing RMS (RH30, RH4) and NB (SH-SY5Y) cells to increasing concentration of mAb46 (0.1–1 μg/ml). Cell lysates were subjected to Western blot analysis using polyclonal antibodies for total and phosphorylated ALK or ERK proteins. Arrowheads indicate membrane-bound (closed) and cytoplasmic (open) full-length ALK kinase, respectively. (B) Time-course analysis of ALK phosphorylation after antibody mAb46 treatment. RH30, SH-SY5Y and RH4 cells were exposed to 1 μg/ml mAb46 for increasing time intervals, and ALK phosphorylation was assessed by Western blot analysis at each time point. Phosphorylation of ERK is also shown. Proteins band density was measured and expressed as fold(s) of control in graphs. Phosphorylated ALK and ERK values were graphed using different scale options. (C) Ligand-independent ALK receptor activity. Relative expression and phosphorylation of ALK RTK was assessed in RH30, SH-SY5Y and RH4 cells after transfection with wild-type (WT) or mutant (F1174L and R1275Q) ALK expression plasmids. Expression and phosphorylation of ALK, ERK, AKT and STAT3 proteins was assessed, using γ-Tubulin as loading control.

Mentions: RTK activation is dynamic process that consists of growth factor binding and receptor autophosphorylation before intracellular signal processing [44]. However, recent observations support the concept that self-association of the extracellular regions of RTKs may occur even in the absence of a specific ligand, particularly in cancer cells in which dimerization takes place spontaneously as a consequence of increased protein expression [45,46,47]. Thus, to look more in detail at the mechanisms of ALK activation, RH30 and SH-SY5Y cells were exposed to increasing amounts of agonist mAb46 antibody and kinetics of receptor phosphorylation was assessed. Antibody treatment led to a marked phosphorylation of ALK in both cell lines, which correlated with a prompt and durable activation of ERK kinase (Fig 2A and 2B). In contrast, mAb46 exposure did not have any effect in RH4 cells, consistent with the scarce expression of ALK in these cells. Therefore, to prove also that receptor density at the plasma membrane could affect intrinsic kinase activity, we transiently transfected RH30, SH-SY5Y and RH4 cells with wild-type or mutant receptor constructs, and assessed protein phosphorylation in the absence of ligand binding. Among the previously identified ALK somatic and germline mutations, we choose hot spot residues F1174L and R1275Q, since mutations at these sites account for more than 70% of mutations in neuroblastoma patients and result in a altered receptor activity [33,48]. Consistent with our hypothesis, overexpression of ALK promoted spontaneous receptor dimerization and activity on the membrane and mediated phosphorylation of downstream targets ERK1/2, AKT and STAT3 independently of its mutational status (Fig 2C) [49]. Perhaps the most striking observation from this analysis was that ALK levels affected RMS cell signaling even in the absence of activating mutations or growth factor binding, establishing a novel genotype-therapeutic correlation that can be used to identify patients who most likely respond to ALK kinase inhibitors.


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)

Kinetics of ALK receptor stimulation and signalling.(A) Antibody-dependent ALK activation was induced by exposing RMS (RH30, RH4) and NB (SH-SY5Y) cells to increasing concentration of mAb46 (0.1–1 μg/ml). Cell lysates were subjected to Western blot analysis using polyclonal antibodies for total and phosphorylated ALK or ERK proteins. Arrowheads indicate membrane-bound (closed) and cytoplasmic (open) full-length ALK kinase, respectively. (B) Time-course analysis of ALK phosphorylation after antibody mAb46 treatment. RH30, SH-SY5Y and RH4 cells were exposed to 1 μg/ml mAb46 for increasing time intervals, and ALK phosphorylation was assessed by Western blot analysis at each time point. Phosphorylation of ERK is also shown. Proteins band density was measured and expressed as fold(s) of control in graphs. Phosphorylated ALK and ERK values were graphed using different scale options. (C) Ligand-independent ALK receptor activity. Relative expression and phosphorylation of ALK RTK was assessed in RH30, SH-SY5Y and RH4 cells after transfection with wild-type (WT) or mutant (F1174L and R1275Q) ALK expression plasmids. Expression and phosphorylation of ALK, ERK, AKT and STAT3 proteins was assessed, using γ-Tubulin as loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132330.g002: Kinetics of ALK receptor stimulation and signalling.(A) Antibody-dependent ALK activation was induced by exposing RMS (RH30, RH4) and NB (SH-SY5Y) cells to increasing concentration of mAb46 (0.1–1 μg/ml). Cell lysates were subjected to Western blot analysis using polyclonal antibodies for total and phosphorylated ALK or ERK proteins. Arrowheads indicate membrane-bound (closed) and cytoplasmic (open) full-length ALK kinase, respectively. (B) Time-course analysis of ALK phosphorylation after antibody mAb46 treatment. RH30, SH-SY5Y and RH4 cells were exposed to 1 μg/ml mAb46 for increasing time intervals, and ALK phosphorylation was assessed by Western blot analysis at each time point. Phosphorylation of ERK is also shown. Proteins band density was measured and expressed as fold(s) of control in graphs. Phosphorylated ALK and ERK values were graphed using different scale options. (C) Ligand-independent ALK receptor activity. Relative expression and phosphorylation of ALK RTK was assessed in RH30, SH-SY5Y and RH4 cells after transfection with wild-type (WT) or mutant (F1174L and R1275Q) ALK expression plasmids. Expression and phosphorylation of ALK, ERK, AKT and STAT3 proteins was assessed, using γ-Tubulin as loading control.
Mentions: RTK activation is dynamic process that consists of growth factor binding and receptor autophosphorylation before intracellular signal processing [44]. However, recent observations support the concept that self-association of the extracellular regions of RTKs may occur even in the absence of a specific ligand, particularly in cancer cells in which dimerization takes place spontaneously as a consequence of increased protein expression [45,46,47]. Thus, to look more in detail at the mechanisms of ALK activation, RH30 and SH-SY5Y cells were exposed to increasing amounts of agonist mAb46 antibody and kinetics of receptor phosphorylation was assessed. Antibody treatment led to a marked phosphorylation of ALK in both cell lines, which correlated with a prompt and durable activation of ERK kinase (Fig 2A and 2B). In contrast, mAb46 exposure did not have any effect in RH4 cells, consistent with the scarce expression of ALK in these cells. Therefore, to prove also that receptor density at the plasma membrane could affect intrinsic kinase activity, we transiently transfected RH30, SH-SY5Y and RH4 cells with wild-type or mutant receptor constructs, and assessed protein phosphorylation in the absence of ligand binding. Among the previously identified ALK somatic and germline mutations, we choose hot spot residues F1174L and R1275Q, since mutations at these sites account for more than 70% of mutations in neuroblastoma patients and result in a altered receptor activity [33,48]. Consistent with our hypothesis, overexpression of ALK promoted spontaneous receptor dimerization and activity on the membrane and mediated phosphorylation of downstream targets ERK1/2, AKT and STAT3 independently of its mutational status (Fig 2C) [49]. Perhaps the most striking observation from this analysis was that ALK levels affected RMS cell signaling even in the absence of activating mutations or growth factor binding, establishing a novel genotype-therapeutic correlation that can be used to identify patients who most likely respond to ALK kinase inhibitors.

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