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Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma

View Article: PubMed Central - PubMed

ABSTRACT

In neuroblastoma (NB), one of the most common paediatric solid tumours, activation of anaplastic lymphoma kinase (ALK) is often associated with poor outcomes. Although genetic studies have identified copy number alteration and nonsynonymous mutations of ALK, the regulatory mechanism of ALK signalling at protein levels is largely elusive. Neuronal leucine-rich repeat 1 (NLRR1) is a type 1 transmembrane protein that is highly expressed in unfavourable NB and potentially influences receptor tyrosine kinase signalling. Here, we showed that NLRR1 and ALK exhibited a mutually exclusive expression pattern in primary NB tissues by immunohistochemistry. Moreover, dorsal root ganglia of Nlrr1+/+ and Nlrr1−/− mice displayed the opposite expression patterns of Nlrr1 and Alk. Of interest, NLRR1 physically interacted with ALK in vitro through its extracellular region. Notably, the NLRR1 ectodomain impaired ALK phosphorylation and proliferation of ALK-mutated NB cells. A newly identified cleavage of the NLRR1 ectodomain also supported NLRR1-mediated ALK signal regulation in trans. Thus, we conclude that NLRR1 appears to be an extracellular negative regulator of ALK signalling in NB and neuronal development. Our findings may be beneficial to comprehend NB heterogeneity and to develop a novel therapy against unfavourable NB.

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NLRR1 impairs ALK-expressing cell expansion.(a) ALK-expressing NB cell expansion was repressed by co-culture with NLRR1-expressing cells. Cell expansion for 5 days was evaluated as the increase of EGFP intensity. Data are means ± s.d. (b) ALK-mutated NB cell growth was inhibited by a fixed layer of NLRR1-expressing cells. (d) exN1 abrogated ALK-mutated NB cell proliferation and the LRR domain was required for the effect. (d) pexN1 reduced SH-SY5Y cell growth. Data are means ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001.
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f5: NLRR1 impairs ALK-expressing cell expansion.(a) ALK-expressing NB cell expansion was repressed by co-culture with NLRR1-expressing cells. Cell expansion for 5 days was evaluated as the increase of EGFP intensity. Data are means ± s.d. (b) ALK-mutated NB cell growth was inhibited by a fixed layer of NLRR1-expressing cells. (d) exN1 abrogated ALK-mutated NB cell proliferation and the LRR domain was required for the effect. (d) pexN1 reduced SH-SY5Y cell growth. Data are means ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001.

Mentions: Considering the negative effect of the NLRR1 ectodomain on ALK signalling, we hypothesised that the proliferation of ALK-mutated NB cells might be suppressed by the NLRR1 ectodomain. In the co-culture assay performed in Fig. 4b, the increase of total EGFP intensity in CHP134-ALK and SH-SY5Y cells was significantly diminished by the neighbouring MCF7-NLRR1 cells (Fig. 5a and Supplementary Figure 4a). The live MCF7-NLRR1 cells possibly occupied a larger area than the control cells, resulting in suppression of NB cell growth in the co-culture condition. Hence, we next fixed MCF7-NLRR1 cells on the surface of the culture plate and then incubated SH-SY5Y and NB-39-nu cells in the wells. Consequently, the cell proliferation was remarkably attenuated by culture on the fixed NLRR1-expressing cell layer compared with that on the fixed control cells (Fig. 5b). To evaluate the inhibitory effect on cell growth by the exN1 molecule, Fc-chimeric exN1 and its deletion mutants were introduced into 96-well plates coated with protein G. Then, SH-SY5Y and NB-39-nu cells were cultured in these plates. Of the chimeric proteins, fixed exN1-Fc suppressed cell proliferation, whereas the other two deletion mutants exhibited marginal effects (Fig. 5c). This result also suggested that the negative effect of NLRR1 on ALK required the LRR domain. Furthermore, pexN1 treatment decreased the growth of SH-SY5Y cells compared with untreated and vehicle-treated controls (Fig. 5d and Supplementary Figure 4b). Therefore, the NLRR1 ectodomain certainly suppressed ALK-mutated NB cell proliferation.


Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma
NLRR1 impairs ALK-expressing cell expansion.(a) ALK-expressing NB cell expansion was repressed by co-culture with NLRR1-expressing cells. Cell expansion for 5 days was evaluated as the increase of EGFP intensity. Data are means ± s.d. (b) ALK-mutated NB cell growth was inhibited by a fixed layer of NLRR1-expressing cells. (d) exN1 abrogated ALK-mutated NB cell proliferation and the LRR domain was required for the effect. (d) pexN1 reduced SH-SY5Y cell growth. Data are means ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015029&req=5

f5: NLRR1 impairs ALK-expressing cell expansion.(a) ALK-expressing NB cell expansion was repressed by co-culture with NLRR1-expressing cells. Cell expansion for 5 days was evaluated as the increase of EGFP intensity. Data are means ± s.d. (b) ALK-mutated NB cell growth was inhibited by a fixed layer of NLRR1-expressing cells. (d) exN1 abrogated ALK-mutated NB cell proliferation and the LRR domain was required for the effect. (d) pexN1 reduced SH-SY5Y cell growth. Data are means ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001.
Mentions: Considering the negative effect of the NLRR1 ectodomain on ALK signalling, we hypothesised that the proliferation of ALK-mutated NB cells might be suppressed by the NLRR1 ectodomain. In the co-culture assay performed in Fig. 4b, the increase of total EGFP intensity in CHP134-ALK and SH-SY5Y cells was significantly diminished by the neighbouring MCF7-NLRR1 cells (Fig. 5a and Supplementary Figure 4a). The live MCF7-NLRR1 cells possibly occupied a larger area than the control cells, resulting in suppression of NB cell growth in the co-culture condition. Hence, we next fixed MCF7-NLRR1 cells on the surface of the culture plate and then incubated SH-SY5Y and NB-39-nu cells in the wells. Consequently, the cell proliferation was remarkably attenuated by culture on the fixed NLRR1-expressing cell layer compared with that on the fixed control cells (Fig. 5b). To evaluate the inhibitory effect on cell growth by the exN1 molecule, Fc-chimeric exN1 and its deletion mutants were introduced into 96-well plates coated with protein G. Then, SH-SY5Y and NB-39-nu cells were cultured in these plates. Of the chimeric proteins, fixed exN1-Fc suppressed cell proliferation, whereas the other two deletion mutants exhibited marginal effects (Fig. 5c). This result also suggested that the negative effect of NLRR1 on ALK required the LRR domain. Furthermore, pexN1 treatment decreased the growth of SH-SY5Y cells compared with untreated and vehicle-treated controls (Fig. 5d and Supplementary Figure 4b). Therefore, the NLRR1 ectodomain certainly suppressed ALK-mutated NB cell proliferation.

View Article: PubMed Central - PubMed

ABSTRACT

In neuroblastoma (NB), one of the most common paediatric solid tumours, activation of anaplastic lymphoma kinase (ALK) is often associated with poor outcomes. Although genetic studies have identified copy number alteration and nonsynonymous mutations of ALK, the regulatory mechanism of ALK signalling at protein levels is largely elusive. Neuronal leucine-rich repeat 1 (NLRR1) is a type 1 transmembrane protein that is highly expressed in unfavourable NB and potentially influences receptor tyrosine kinase signalling. Here, we showed that NLRR1 and ALK exhibited a mutually exclusive expression pattern in primary NB tissues by immunohistochemistry. Moreover, dorsal root ganglia of Nlrr1+/+ and Nlrr1&minus;/&minus; mice displayed the opposite expression patterns of Nlrr1 and Alk. Of interest, NLRR1 physically interacted with ALK in vitro through its extracellular region. Notably, the NLRR1 ectodomain impaired ALK phosphorylation and proliferation of ALK-mutated NB cells. A newly identified cleavage of the NLRR1 ectodomain also supported NLRR1-mediated ALK signal regulation in trans. Thus, we conclude that NLRR1 appears to be an extracellular negative regulator of ALK signalling in NB and neuronal development. Our findings may be beneficial to comprehend NB heterogeneity and to develop a novel therapy against unfavourable NB.

No MeSH data available.


Related in: MedlinePlus