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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

NGF induces association of TrkA with CD44/p115RhoGEF/RhoA/RhoC/ROCK1 and RhoGTPase activation(A–D) NGF induces TrkA association with CD44/p115RhoGEF/RhoGTPases/ROCK1. HA-TrkA MDA-MB-231 cells were treated with NGF (5 and 30 min). IPs were done with anti-HA or anti-CD44 antibodies, and immunoblotting was used to detect the presence of p115RhoGEF (A and B) and ROCK1 (C and D) in the eluate. (E) NGF increases RhoGTPase activity. HA-TrkA MDA-MB-231 cells were stimulated with NGF (5 or 30 min) and GTP-bound RhoA and RhoC were determined in cell lysates by an affinity pull-down assay with the GST-Rho binding domain followed by immunoblotting for RhoA and RhoC. Whole cell lysate samples were immunoblotted for total RhoA/C as a control. Figures are representative of three independent pull-down assays. (F) CD44 invalidation inhibits RhoA and RhoC activities. HA-TrkA MDA-MB-231 cells were transfected with siCTRL (scramble siRNA) or siCD44 stimulated with NGF (5 or 30 min) and GTP-bound RhoA and RhoC were determined in cell lysates by an affinity pull-down assay with the GST-Rho binding domain followed by immunoblotting for RhoA and RhoC. Whole cell lysate samples were immunoblotted for total RhoA/C as a control.
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Figure 2: NGF induces association of TrkA with CD44/p115RhoGEF/RhoA/RhoC/ROCK1 and RhoGTPase activation(A–D) NGF induces TrkA association with CD44/p115RhoGEF/RhoGTPases/ROCK1. HA-TrkA MDA-MB-231 cells were treated with NGF (5 and 30 min). IPs were done with anti-HA or anti-CD44 antibodies, and immunoblotting was used to detect the presence of p115RhoGEF (A and B) and ROCK1 (C and D) in the eluate. (E) NGF increases RhoGTPase activity. HA-TrkA MDA-MB-231 cells were stimulated with NGF (5 or 30 min) and GTP-bound RhoA and RhoC were determined in cell lysates by an affinity pull-down assay with the GST-Rho binding domain followed by immunoblotting for RhoA and RhoC. Whole cell lysate samples were immunoblotted for total RhoA/C as a control. Figures are representative of three independent pull-down assays. (F) CD44 invalidation inhibits RhoA and RhoC activities. HA-TrkA MDA-MB-231 cells were transfected with siCTRL (scramble siRNA) or siCD44 stimulated with NGF (5 or 30 min) and GTP-bound RhoA and RhoC were determined in cell lysates by an affinity pull-down assay with the GST-Rho binding domain followed by immunoblotting for RhoA and RhoC. Whole cell lysate samples were immunoblotted for total RhoA/C as a control.

Mentions: CD44 is known to stimulate breast tumor cell invasion through the RhoA pathway [20]. Interestingly, we found that p115RhoGEF co-immunoprecipitated with TrkA in NGF-treated cells (Table 1). To confirm the downstream association of p115RhoGEF to the TrkA/CD44 complex, we used co-IP to investigate whether they directly interact. Indeed, antibodies against the HA tag or CD44 were able to pull down p115RhoGEF (Figure 2A and 2B). Moreover, upon NGF treatment, ROCK1 (the main target of RhoA and RhoC) was also found to co-IP with TrkA and CD44 (Figure 2C and 2D). The involvement of p115RhoGEF and ROCK1 in NGF-induced signaling was further supported by the activation of RhoA and RhoC, which are p115RhoGEF downstream targets (Figure 2E). In order to ascertain that CD44 is responsible of RhoGTPase activation, we measured RhoA and RhoC activities after CD44 invalidation by siRNA. As shown in Figure 2F, CD44 siRNA reduced RhoGTPAse activities for both RhoA and RhoC. We also analyzed the invasive capacity of breast cancer cells in vitro using a transwell system (Figure 3A–3C). We found that inhibition of CD44 and p115RhoGEF either through siRNA or the ROCK1 specific inhibitor Y-27632 totally abolished NGF-induced invasion Interestingly, blocking CD44 affected neither NGF-induced TrkA phosphorylation nor the canonical TrkA pathways that signal through Akt and Src (Supplementary Figure S3). Together, these results indicate that the TrkA/CD44 complex induced by NGF is able to activate the RhoGTPAse signaling pathway to enhance cell invasion, independently of TrkA canonical pathways.


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)

NGF induces association of TrkA with CD44/p115RhoGEF/RhoA/RhoC/ROCK1 and RhoGTPase activation(A–D) NGF induces TrkA association with CD44/p115RhoGEF/RhoGTPases/ROCK1. HA-TrkA MDA-MB-231 cells were treated with NGF (5 and 30 min). IPs were done with anti-HA or anti-CD44 antibodies, and immunoblotting was used to detect the presence of p115RhoGEF (A and B) and ROCK1 (C and D) in the eluate. (E) NGF increases RhoGTPase activity. HA-TrkA MDA-MB-231 cells were stimulated with NGF (5 or 30 min) and GTP-bound RhoA and RhoC were determined in cell lysates by an affinity pull-down assay with the GST-Rho binding domain followed by immunoblotting for RhoA and RhoC. Whole cell lysate samples were immunoblotted for total RhoA/C as a control. Figures are representative of three independent pull-down assays. (F) CD44 invalidation inhibits RhoA and RhoC activities. HA-TrkA MDA-MB-231 cells were transfected with siCTRL (scramble siRNA) or siCD44 stimulated with NGF (5 or 30 min) and GTP-bound RhoA and RhoC were determined in cell lysates by an affinity pull-down assay with the GST-Rho binding domain followed by immunoblotting for RhoA and RhoC. Whole cell lysate samples were immunoblotted for total RhoA/C as a control.
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Related In: Results  -  Collection

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Figure 2: NGF induces association of TrkA with CD44/p115RhoGEF/RhoA/RhoC/ROCK1 and RhoGTPase activation(A–D) NGF induces TrkA association with CD44/p115RhoGEF/RhoGTPases/ROCK1. HA-TrkA MDA-MB-231 cells were treated with NGF (5 and 30 min). IPs were done with anti-HA or anti-CD44 antibodies, and immunoblotting was used to detect the presence of p115RhoGEF (A and B) and ROCK1 (C and D) in the eluate. (E) NGF increases RhoGTPase activity. HA-TrkA MDA-MB-231 cells were stimulated with NGF (5 or 30 min) and GTP-bound RhoA and RhoC were determined in cell lysates by an affinity pull-down assay with the GST-Rho binding domain followed by immunoblotting for RhoA and RhoC. Whole cell lysate samples were immunoblotted for total RhoA/C as a control. Figures are representative of three independent pull-down assays. (F) CD44 invalidation inhibits RhoA and RhoC activities. HA-TrkA MDA-MB-231 cells were transfected with siCTRL (scramble siRNA) or siCD44 stimulated with NGF (5 or 30 min) and GTP-bound RhoA and RhoC were determined in cell lysates by an affinity pull-down assay with the GST-Rho binding domain followed by immunoblotting for RhoA and RhoC. Whole cell lysate samples were immunoblotted for total RhoA/C as a control.
Mentions: CD44 is known to stimulate breast tumor cell invasion through the RhoA pathway [20]. Interestingly, we found that p115RhoGEF co-immunoprecipitated with TrkA in NGF-treated cells (Table 1). To confirm the downstream association of p115RhoGEF to the TrkA/CD44 complex, we used co-IP to investigate whether they directly interact. Indeed, antibodies against the HA tag or CD44 were able to pull down p115RhoGEF (Figure 2A and 2B). Moreover, upon NGF treatment, ROCK1 (the main target of RhoA and RhoC) was also found to co-IP with TrkA and CD44 (Figure 2C and 2D). The involvement of p115RhoGEF and ROCK1 in NGF-induced signaling was further supported by the activation of RhoA and RhoC, which are p115RhoGEF downstream targets (Figure 2E). In order to ascertain that CD44 is responsible of RhoGTPase activation, we measured RhoA and RhoC activities after CD44 invalidation by siRNA. As shown in Figure 2F, CD44 siRNA reduced RhoGTPAse activities for both RhoA and RhoC. We also analyzed the invasive capacity of breast cancer cells in vitro using a transwell system (Figure 3A–3C). We found that inhibition of CD44 and p115RhoGEF either through siRNA or the ROCK1 specific inhibitor Y-27632 totally abolished NGF-induced invasion Interestingly, blocking CD44 affected neither NGF-induced TrkA phosphorylation nor the canonical TrkA pathways that signal through Akt and Src (Supplementary Figure S3). Together, these results indicate that the TrkA/CD44 complex induced by NGF is able to activate the RhoGTPAse signaling pathway to enhance cell invasion, independently of TrkA canonical pathways.

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