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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

RMS cell growth and signalling is downregulated by ALK inhibitors.(A) Cell cycle analysis of RMS cells treated with the indicated doses of Crizotinib and TAE684 for 24 hours. Cell cycle phases distribution measured with FACS-Calibur Cell Cytometer and represented by stacked bar graphs. The percentage of cells in sub G1 phase is shown. (B) Lysates from ALK inhibitor-treated and untreated RMS cells, probed with antibodies directed against total and phosphorylated AKT and ERK proteins, as well as against full-length and cleaved PARP (arrowhead). γ-Tubulin was used as internal loading control.
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pone.0132330.g005: RMS cell growth and signalling is downregulated by ALK inhibitors.(A) Cell cycle analysis of RMS cells treated with the indicated doses of Crizotinib and TAE684 for 24 hours. Cell cycle phases distribution measured with FACS-Calibur Cell Cytometer and represented by stacked bar graphs. The percentage of cells in sub G1 phase is shown. (B) Lysates from ALK inhibitor-treated and untreated RMS cells, probed with antibodies directed against total and phosphorylated AKT and ERK proteins, as well as against full-length and cleaved PARP (arrowhead). γ-Tubulin was used as internal loading control.

Mentions: Almost all protein kinases share the same conserved sequences around the ATP-binding site, as they all have the same phosphotransferase activity toward common or uncommon protein substrates [52]. Small molecules targeting the ATP binding cleft may have, thus, an inherent multi-target nature and be active in cells where the primary target is weakly expressed or even absent [53,54]. Consistent with these findings, we found that crizotinib inhibited basal ERK phosphorylation in RMS cells independently of ALK expression and activity, as this phenomenon was observed in RH4 cells as well (Fig 2C). Therefore, we tested the ability of ALK inhibitors to impede the growth of ALK-positive and–negative RMS cells, when administered at increasing concentrations and prolonged time intervals. Crizotinib was chosen for its dual ALK/Met inhibitor activity, while NPV-TAE684 (TAE684) for its affinity for ALK and, to a lesser extent, IGF-1R [55,56]. We found that at low doses TAE684 showed stronger inhibitory potency than crizotinib, whereas at higher concentrations such difference was less significant and not associated with ALK expression (Fig 4). Conversely, transformed cells overexpressing full-length (NB1) or truncated (SU-DHL-1) ALK kinase displayed a much higher sensitivity compared to RMS cells, consistent with the superior dependency of these cells on ALK signaling. Moreover, when crizotinib and TAE684 were administered for shorter time intervals, all RMS cell lines exhibited comparable cell cycle arrest (at G1 or G2/M phase) and apoptosis (PARP cleavage), which correlated with downregulation of PI3K-AKT (pAKT) and MAPK (pERK) survival signaling pathways (Fig 5A and 5B). In line with these observations, constitutive ligand-independent activation of amplified c-Met receptor is used as molecular marker of susceptibility to tyrosine kinase inhibitors in human gastric cancer cell lines, since only cells exhibiting high-level expression of wild-type c-Met appear to be sensitive to Met inhibition by drug treatment [57,58].


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)

RMS cell growth and signalling is downregulated by ALK inhibitors.(A) Cell cycle analysis of RMS cells treated with the indicated doses of Crizotinib and TAE684 for 24 hours. Cell cycle phases distribution measured with FACS-Calibur Cell Cytometer and represented by stacked bar graphs. The percentage of cells in sub G1 phase is shown. (B) Lysates from ALK inhibitor-treated and untreated RMS cells, probed with antibodies directed against total and phosphorylated AKT and ERK proteins, as well as against full-length and cleaved PARP (arrowhead). γ-Tubulin was used as internal loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132330.g005: RMS cell growth and signalling is downregulated by ALK inhibitors.(A) Cell cycle analysis of RMS cells treated with the indicated doses of Crizotinib and TAE684 for 24 hours. Cell cycle phases distribution measured with FACS-Calibur Cell Cytometer and represented by stacked bar graphs. The percentage of cells in sub G1 phase is shown. (B) Lysates from ALK inhibitor-treated and untreated RMS cells, probed with antibodies directed against total and phosphorylated AKT and ERK proteins, as well as against full-length and cleaved PARP (arrowhead). γ-Tubulin was used as internal loading control.
Mentions: Almost all protein kinases share the same conserved sequences around the ATP-binding site, as they all have the same phosphotransferase activity toward common or uncommon protein substrates [52]. Small molecules targeting the ATP binding cleft may have, thus, an inherent multi-target nature and be active in cells where the primary target is weakly expressed or even absent [53,54]. Consistent with these findings, we found that crizotinib inhibited basal ERK phosphorylation in RMS cells independently of ALK expression and activity, as this phenomenon was observed in RH4 cells as well (Fig 2C). Therefore, we tested the ability of ALK inhibitors to impede the growth of ALK-positive and–negative RMS cells, when administered at increasing concentrations and prolonged time intervals. Crizotinib was chosen for its dual ALK/Met inhibitor activity, while NPV-TAE684 (TAE684) for its affinity for ALK and, to a lesser extent, IGF-1R [55,56]. We found that at low doses TAE684 showed stronger inhibitory potency than crizotinib, whereas at higher concentrations such difference was less significant and not associated with ALK expression (Fig 4). Conversely, transformed cells overexpressing full-length (NB1) or truncated (SU-DHL-1) ALK kinase displayed a much higher sensitivity compared to RMS cells, consistent with the superior dependency of these cells on ALK signaling. Moreover, when crizotinib and TAE684 were administered for shorter time intervals, all RMS cell lines exhibited comparable cell cycle arrest (at G1 or G2/M phase) and apoptosis (PARP cleavage), which correlated with downregulation of PI3K-AKT (pAKT) and MAPK (pERK) survival signaling pathways (Fig 5A and 5B). In line with these observations, constitutive ligand-independent activation of amplified c-Met receptor is used as molecular marker of susceptibility to tyrosine kinase inhibitors in human gastric cancer cell lines, since only cells exhibiting high-level expression of wild-type c-Met appear to be sensitive to Met inhibition by drug treatment [57,58].

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