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NGF-induced TrkA/CD44 association is involved in tumor aggressiveness and resistance to lestaurtinib.

Aubert L, Guilbert M, Corbet C, Génot E, Adriaenssens E, Chassat T, Bertucci F, Daubon T, Magné N, Le Bourhis X, Toillon RA - Oncotarget (2015)

Bottom Line: We found that NGF treatment induced CD44 binding to TrkA at the plasma membrane and subsequent activation of the p115RhoGEF/RhoA/ROCK1 pathway to stimulate breast cancer cell invasion.Moreover, both TrkA tyrosine kinase inhibition with lestaurtinib and CD44 silencing with siRNA inhibited cell growth in vitro as well as tumor development in mouse xenograft model; combined treatment significantly enhanced the antineoplastic effects of either treatment alone.Altogether, our results demonstrate that NGF-induced tyrosine kinase independent TrkA signaling through CD44 was sufficient to maintain tumor aggressiveness.

View Article: PubMed Central - PubMed

Affiliation: INSERM U908, 59655 Villeneuve d'Ascq, France.

ABSTRACT
There is accumulating evidence that TrkA and its ligand Nerve Growth Factor (NGF) are involved in cancer development. Staurosporine derivatives such as K252a and lestaurtinib have been developed to block TrkA kinase signaling, but no clinical trial has fully demonstrated their therapeutic efficacy. Therapeutic failures are likely due to the existence of intrinsic signaling pathways in cancer cells that impede or bypass the effects of TrkA tyrosine kinase inhibitors. To verify this hypothesis, we combined different approaches including mass spectrometry proteomics, co-immunoprecipitation and proximity ligation assays. We found that NGF treatment induced CD44 binding to TrkA at the plasma membrane and subsequent activation of the p115RhoGEF/RhoA/ROCK1 pathway to stimulate breast cancer cell invasion. The NGF-induced CD44 signaling was independent of TrkA kinase activity. Moreover, both TrkA tyrosine kinase inhibition with lestaurtinib and CD44 silencing with siRNA inhibited cell growth in vitro as well as tumor development in mouse xenograft model; combined treatment significantly enhanced the antineoplastic effects of either treatment alone. Altogether, our results demonstrate that NGF-induced tyrosine kinase independent TrkA signaling through CD44 was sufficient to maintain tumor aggressiveness. Our findings provide an alternative mechanism of cancer resistance to lestaurtinib and indicate that dual inhibition of CD44 and TrkA tyrosine kinase activity may represent a novel therapeutic strategy.

No MeSH data available.


Related in: MedlinePlus

Proposed model of lestaurtinib resistance through a phospho-TrkA-independent pathway involving downstream CD44 signalingLestaurtinib inhibits the kinase activity of TrkA and phospho-TrkA-dependent downstream signaling including Akt. However, a phospho-TrkA-independent pathway that uses CD44 signaling may serve as an alternative pathway to strengthen tumor aggressiveness and to escape lestaurtinib inhibition.
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Figure 6: Proposed model of lestaurtinib resistance through a phospho-TrkA-independent pathway involving downstream CD44 signalingLestaurtinib inhibits the kinase activity of TrkA and phospho-TrkA-dependent downstream signaling including Akt. However, a phospho-TrkA-independent pathway that uses CD44 signaling may serve as an alternative pathway to strengthen tumor aggressiveness and to escape lestaurtinib inhibition.

Mentions: In this study, we report for the first time that TrkA is associated with CD44 in cancer cells. CD44 is the principal receptor for the large glycosaminoglycan hyaluronan. CD44 lacks kinase activity but influences cell behaviors such as uncontrolled growth, apoptosis evasion, angiogenesis, cell motility and invasion through various mechanisms [22]. CD44 directly interacts with key intracellular regulators of the actin cytoskeleton such as ankyrin, and members of the ezrin, radixin, and moesin (ERM) protein families. CD44 can also function as a co-receptor to modulate signaling from a diverse set of membrane receptors including integrins, multidrug resistance complex components (MDR 1, CD147) and growth factor receptors such as EGFR, PDGFR, FGFR, and c-Met [19]. CD44 cooperates with RTKs through different mechanisms. For instance, CD44 transactivates the ErbB2/ErbB3 heterodimer by Src-mediated phosphorylation of ErbB2. CD44 can also initiate growth factor receptor signaling by interacting with various ligands. For instance, binding of proHBEGF to CD44v3 induces cleavage of the proform and release of HBEGF that in turn activates ErbB4. CD44v also binds to HGF/SF and FGF and presents them to their specific RTK [23]. A splice variant of CD44 expressed in the apical ectoderm ridge presents FGFs to limb mesenchyme and is required for limb outgrowth [24]. In our cell models, because CD44 is co-immunoprecipitated with TrkA and we observed a positive signal from the PLA, a direct association between TrkA and CD44 is suggested. Alternatively, NGF is known to bind to heparan sulfate with low affinity [25], it is possible that NGF serves as a bridge between TrkA and CD44. Moreover, the formation of TrkA/CD44 complex upon NGF stimulation leads to the activation of the canonical CD44 pathway involving p115RhoGEF, RhoA and ROCK1 (Figure 6). TrkA and CD44 co-expression has been already reported in neuroblastoma cells [26] but the authors did not examine their interaction nor the resulting intracellular signaling. TrkA signaling has been widely described in nerve systems and similar signaling pathways have also been found in cancer cells. Indeed, NGF binding to TrkA induces phosphorylation of TrkA, phospho-TrkA then recruits various intracellular adaptors (e.g. Shc, Grb2, or PI3K) to activate MAPK or PI3K, resulting in cell growth or differentiation depending on the cell type. TrkA signaling is also modulated by several co-receptors such as p75NTR, GPCR (G protein-coupled receptor), and Ret-5. P75NTR is a low affinity receptor and is the first described co-receptor of TrkA. After its binding to p75NTR, NGF has an increased affinity for TrkA [27]. In addition, p75NTR also delays TrkA ubiquitination and sustains its phosphorylation [28]. However, we have previously shown that p75NTR is not involved in NGF-stimulated invasion of breast cancer cells [8]. Transactivation of TrkA receptors in PC12 cells and TrkB in hippocampal neurons has been observed after treatment with adenosine or PACAP neuromodulators, both of which act through GPCRs [29]. Trk receptor transactivation by adenosine or PACAP requires a longer time course. Furthermore, the increase in Trk activity can be inhibited by the use of K252a, PP1 and Src family-specific inhibitors. Trk receptors also have the capacity to activate the Ret-51 receptor tyrosine kinase in postnatal superior cervical ganglion (SCG) neurons. Ret-51 activation does not depend on the PI3K or MAPK pathways and occurs with a very slow kinetics, similar to GPCR activation of Trk receptors [30].


NGF-induced TrkA/CD44 association is involved in tumor aggressiveness and resistance to lestaurtinib.

Aubert L, Guilbert M, Corbet C, Génot E, Adriaenssens E, Chassat T, Bertucci F, Daubon T, Magné N, Le Bourhis X, Toillon RA - Oncotarget (2015)

Proposed model of lestaurtinib resistance through a phospho-TrkA-independent pathway involving downstream CD44 signalingLestaurtinib inhibits the kinase activity of TrkA and phospho-TrkA-dependent downstream signaling including Akt. However, a phospho-TrkA-independent pathway that uses CD44 signaling may serve as an alternative pathway to strengthen tumor aggressiveness and to escape lestaurtinib inhibition.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496399&req=5

Figure 6: Proposed model of lestaurtinib resistance through a phospho-TrkA-independent pathway involving downstream CD44 signalingLestaurtinib inhibits the kinase activity of TrkA and phospho-TrkA-dependent downstream signaling including Akt. However, a phospho-TrkA-independent pathway that uses CD44 signaling may serve as an alternative pathway to strengthen tumor aggressiveness and to escape lestaurtinib inhibition.
Mentions: In this study, we report for the first time that TrkA is associated with CD44 in cancer cells. CD44 is the principal receptor for the large glycosaminoglycan hyaluronan. CD44 lacks kinase activity but influences cell behaviors such as uncontrolled growth, apoptosis evasion, angiogenesis, cell motility and invasion through various mechanisms [22]. CD44 directly interacts with key intracellular regulators of the actin cytoskeleton such as ankyrin, and members of the ezrin, radixin, and moesin (ERM) protein families. CD44 can also function as a co-receptor to modulate signaling from a diverse set of membrane receptors including integrins, multidrug resistance complex components (MDR 1, CD147) and growth factor receptors such as EGFR, PDGFR, FGFR, and c-Met [19]. CD44 cooperates with RTKs through different mechanisms. For instance, CD44 transactivates the ErbB2/ErbB3 heterodimer by Src-mediated phosphorylation of ErbB2. CD44 can also initiate growth factor receptor signaling by interacting with various ligands. For instance, binding of proHBEGF to CD44v3 induces cleavage of the proform and release of HBEGF that in turn activates ErbB4. CD44v also binds to HGF/SF and FGF and presents them to their specific RTK [23]. A splice variant of CD44 expressed in the apical ectoderm ridge presents FGFs to limb mesenchyme and is required for limb outgrowth [24]. In our cell models, because CD44 is co-immunoprecipitated with TrkA and we observed a positive signal from the PLA, a direct association between TrkA and CD44 is suggested. Alternatively, NGF is known to bind to heparan sulfate with low affinity [25], it is possible that NGF serves as a bridge between TrkA and CD44. Moreover, the formation of TrkA/CD44 complex upon NGF stimulation leads to the activation of the canonical CD44 pathway involving p115RhoGEF, RhoA and ROCK1 (Figure 6). TrkA and CD44 co-expression has been already reported in neuroblastoma cells [26] but the authors did not examine their interaction nor the resulting intracellular signaling. TrkA signaling has been widely described in nerve systems and similar signaling pathways have also been found in cancer cells. Indeed, NGF binding to TrkA induces phosphorylation of TrkA, phospho-TrkA then recruits various intracellular adaptors (e.g. Shc, Grb2, or PI3K) to activate MAPK or PI3K, resulting in cell growth or differentiation depending on the cell type. TrkA signaling is also modulated by several co-receptors such as p75NTR, GPCR (G protein-coupled receptor), and Ret-5. P75NTR is a low affinity receptor and is the first described co-receptor of TrkA. After its binding to p75NTR, NGF has an increased affinity for TrkA [27]. In addition, p75NTR also delays TrkA ubiquitination and sustains its phosphorylation [28]. However, we have previously shown that p75NTR is not involved in NGF-stimulated invasion of breast cancer cells [8]. Transactivation of TrkA receptors in PC12 cells and TrkB in hippocampal neurons has been observed after treatment with adenosine or PACAP neuromodulators, both of which act through GPCRs [29]. Trk receptor transactivation by adenosine or PACAP requires a longer time course. Furthermore, the increase in Trk activity can be inhibited by the use of K252a, PP1 and Src family-specific inhibitors. Trk receptors also have the capacity to activate the Ret-51 receptor tyrosine kinase in postnatal superior cervical ganglion (SCG) neurons. Ret-51 activation does not depend on the PI3K or MAPK pathways and occurs with a very slow kinetics, similar to GPCR activation of Trk receptors [30].

Bottom Line: We found that NGF treatment induced CD44 binding to TrkA at the plasma membrane and subsequent activation of the p115RhoGEF/RhoA/ROCK1 pathway to stimulate breast cancer cell invasion.Moreover, both TrkA tyrosine kinase inhibition with lestaurtinib and CD44 silencing with siRNA inhibited cell growth in vitro as well as tumor development in mouse xenograft model; combined treatment significantly enhanced the antineoplastic effects of either treatment alone.Altogether, our results demonstrate that NGF-induced tyrosine kinase independent TrkA signaling through CD44 was sufficient to maintain tumor aggressiveness.

View Article: PubMed Central - PubMed

Affiliation: INSERM U908, 59655 Villeneuve d'Ascq, France.

ABSTRACT
There is accumulating evidence that TrkA and its ligand Nerve Growth Factor (NGF) are involved in cancer development. Staurosporine derivatives such as K252a and lestaurtinib have been developed to block TrkA kinase signaling, but no clinical trial has fully demonstrated their therapeutic efficacy. Therapeutic failures are likely due to the existence of intrinsic signaling pathways in cancer cells that impede or bypass the effects of TrkA tyrosine kinase inhibitors. To verify this hypothesis, we combined different approaches including mass spectrometry proteomics, co-immunoprecipitation and proximity ligation assays. We found that NGF treatment induced CD44 binding to TrkA at the plasma membrane and subsequent activation of the p115RhoGEF/RhoA/ROCK1 pathway to stimulate breast cancer cell invasion. The NGF-induced CD44 signaling was independent of TrkA kinase activity. Moreover, both TrkA tyrosine kinase inhibition with lestaurtinib and CD44 silencing with siRNA inhibited cell growth in vitro as well as tumor development in mouse xenograft model; combined treatment significantly enhanced the antineoplastic effects of either treatment alone. Altogether, our results demonstrate that NGF-induced tyrosine kinase independent TrkA signaling through CD44 was sufficient to maintain tumor aggressiveness. Our findings provide an alternative mechanism of cancer resistance to lestaurtinib and indicate that dual inhibition of CD44 and TrkA tyrosine kinase activity may represent a novel therapeutic strategy.

No MeSH data available.


Related in: MedlinePlus