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

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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 and ALK show mutually exclusive expression in human NB.(a) Expression patterns of NLRR1 and ALK were often mutually exclusive in immunohistochemical analysis. Black and white circles indicate NLRR1-rich/ALK-poor and NLRR1-poor/ALK-rich cancer cell clusters, respectively. Bars: 100 μm. (b) The expression level of NLRR1 was not correlated with that of ALK in qPCR analysis. The correlation was evaluated by Pearson’s test.
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f1: NLRR1 and ALK show mutually exclusive expression in human NB.(a) Expression patterns of NLRR1 and ALK were often mutually exclusive in immunohistochemical analysis. Black and white circles indicate NLRR1-rich/ALK-poor and NLRR1-poor/ALK-rich cancer cell clusters, respectively. Bars: 100 μm. (b) The expression level of NLRR1 was not correlated with that of ALK in qPCR analysis. The correlation was evaluated by Pearson’s test.

Mentions: The expression levels of NLRR1 and ALK are associated with unfavourable outcomes of NB31323334. Hence, we randomly selected six primary tumour samples, which were diagnosed as stage 3 or 4, and then immunohistochemically investigated the expression patterns of NLRR1 and ALK in the specimens. Sample #1 with high expression of NLRR1 was negative for ALK, whereas sample #2 with high expression of ALK exhibited low expression of NLRR1 (Fig. 1a). Notably, three specimens showed intratumoural heterogeneity of NLRR1 and ALK. In samples #3 and #4, NLRR1-rich loci were faintly stained for ALK (Fig. 1a, upper panels of samples #3 and #4), while NLRR1-poor regions displayed higher expression of ALK (Fig. 1a, lower panels of samples #3 and #4). Of interest, NLRR1-rich/ALK-poor and NLRR1-poor/ALK-rich cancer cell clusters were mosaically localised in sample #5 (black and white circles in Fig. 1a, respectively). Although sample #6 did not show the same pattern, NLRR1 and ALK were generally expressed in different cancer cells that displayed separated or neighboured clusters even in an identical tumour. To confirm the heterogeneous expression patterns of NLRR1 and ALK, we stained a commercially available human tissue array including 10 NBs. The results are summarised in Supplementary Table 1. Of the tissue array samples, two tumours with high expression of NLRR1 were indistinctly stained for ALK (Supplementary Figure 1a i–iv). Conversely, three tumours with high expression of ALK rarely harboured NLRR1 (Supplementary Figure 1a vii–x), although one section expressed intermediate levels of NLRR1 and ALK (Supplementary Figure 1a v and vi). Therefore, NLRR1 and ALK tended to be oppositely expressed among different tumours. To reveal the expressional correlation at the mRNA level, we performed qPCR analysis using 87 human NB cDNA samples including all International Neuroblastoma Staging System (INSS) stages with MYCN-amplified and non-amplified tumours. Despite the opposite expression pattern of NLRR1 and ALK in immunohistochemical analyses, NLRR1 and ALK showed no significant correlation (Pearson’s r = 0.035, P = 0.749, Fig. 1b and Supplementary Table 2). Furthermore, in risk factor classifications (MYCN status, INSS, age, histology, primary tumour site, TrkA expression, and prognosis), the expression level of NLRR1 was not associated with that of ALK (Supplementary Table 2). Because a large tumour sample often included both NLRR1-rich/ALK-poor and NLRR1-poor/ALK-rich cancer cells (Fig. 1a, samples #3, #4, and #5), the opposite expression pattern of NLRR1 and ALK might be masked in the homogenised mRNA samples. We further examined the expression pattern of NLRR1 and ALK in human NB cell lines SK-N-BE, SK-N-DZ, CHP134, SMS-SAN, SH-SY5Y, Kelly, and NB-39-nu. Consequently, in both qPCR and western blot analyses, CHP134 and SMS-SAN cells highly expressed NLRR1, but they exhibited relatively low expression of ALK (Supplementary Figure 1b). The expression status might result in no phosphorylation of ALK in SMS-SAN cells that harbour the F1174L mutation. In contrast, other ALK-mutated cell lines, SH-SY5Y (F1174L), Kelly (F1174L), and NB-39-nu (amplified), highly expressed ALK with constitutive activation, but they showed low expression of NLRR1 (Supplementary Figure 1b). The homogenous properties of cultured cell lines might result in the opposite expression patterns of NLRR1 and ALK at the mRNA level, which were not confirmed in clinical samples, although they exhibited no statistical significance. Taken together, NLRR1 and ALK were generally expressed in different populations of NB cells and exhibited mutually exclusive expression patterns mainly in immunohistochemical analyses.


Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma
NLRR1 and ALK show mutually exclusive expression in human NB.(a) Expression patterns of NLRR1 and ALK were often mutually exclusive in immunohistochemical analysis. Black and white circles indicate NLRR1-rich/ALK-poor and NLRR1-poor/ALK-rich cancer cell clusters, respectively. Bars: 100 μm. (b) The expression level of NLRR1 was not correlated with that of ALK in qPCR analysis. The correlation was evaluated by Pearson’s test.
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Related In: Results  -  Collection

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f1: NLRR1 and ALK show mutually exclusive expression in human NB.(a) Expression patterns of NLRR1 and ALK were often mutually exclusive in immunohistochemical analysis. Black and white circles indicate NLRR1-rich/ALK-poor and NLRR1-poor/ALK-rich cancer cell clusters, respectively. Bars: 100 μm. (b) The expression level of NLRR1 was not correlated with that of ALK in qPCR analysis. The correlation was evaluated by Pearson’s test.
Mentions: The expression levels of NLRR1 and ALK are associated with unfavourable outcomes of NB31323334. Hence, we randomly selected six primary tumour samples, which were diagnosed as stage 3 or 4, and then immunohistochemically investigated the expression patterns of NLRR1 and ALK in the specimens. Sample #1 with high expression of NLRR1 was negative for ALK, whereas sample #2 with high expression of ALK exhibited low expression of NLRR1 (Fig. 1a). Notably, three specimens showed intratumoural heterogeneity of NLRR1 and ALK. In samples #3 and #4, NLRR1-rich loci were faintly stained for ALK (Fig. 1a, upper panels of samples #3 and #4), while NLRR1-poor regions displayed higher expression of ALK (Fig. 1a, lower panels of samples #3 and #4). Of interest, NLRR1-rich/ALK-poor and NLRR1-poor/ALK-rich cancer cell clusters were mosaically localised in sample #5 (black and white circles in Fig. 1a, respectively). Although sample #6 did not show the same pattern, NLRR1 and ALK were generally expressed in different cancer cells that displayed separated or neighboured clusters even in an identical tumour. To confirm the heterogeneous expression patterns of NLRR1 and ALK, we stained a commercially available human tissue array including 10 NBs. The results are summarised in Supplementary Table 1. Of the tissue array samples, two tumours with high expression of NLRR1 were indistinctly stained for ALK (Supplementary Figure 1a i–iv). Conversely, three tumours with high expression of ALK rarely harboured NLRR1 (Supplementary Figure 1a vii–x), although one section expressed intermediate levels of NLRR1 and ALK (Supplementary Figure 1a v and vi). Therefore, NLRR1 and ALK tended to be oppositely expressed among different tumours. To reveal the expressional correlation at the mRNA level, we performed qPCR analysis using 87 human NB cDNA samples including all International Neuroblastoma Staging System (INSS) stages with MYCN-amplified and non-amplified tumours. Despite the opposite expression pattern of NLRR1 and ALK in immunohistochemical analyses, NLRR1 and ALK showed no significant correlation (Pearson’s r = 0.035, P = 0.749, Fig. 1b and Supplementary Table 2). Furthermore, in risk factor classifications (MYCN status, INSS, age, histology, primary tumour site, TrkA expression, and prognosis), the expression level of NLRR1 was not associated with that of ALK (Supplementary Table 2). Because a large tumour sample often included both NLRR1-rich/ALK-poor and NLRR1-poor/ALK-rich cancer cells (Fig. 1a, samples #3, #4, and #5), the opposite expression pattern of NLRR1 and ALK might be masked in the homogenised mRNA samples. We further examined the expression pattern of NLRR1 and ALK in human NB cell lines SK-N-BE, SK-N-DZ, CHP134, SMS-SAN, SH-SY5Y, Kelly, and NB-39-nu. Consequently, in both qPCR and western blot analyses, CHP134 and SMS-SAN cells highly expressed NLRR1, but they exhibited relatively low expression of ALK (Supplementary Figure 1b). The expression status might result in no phosphorylation of ALK in SMS-SAN cells that harbour the F1174L mutation. In contrast, other ALK-mutated cell lines, SH-SY5Y (F1174L), Kelly (F1174L), and NB-39-nu (amplified), highly expressed ALK with constitutive activation, but they showed low expression of NLRR1 (Supplementary Figure 1b). The homogenous properties of cultured cell lines might result in the opposite expression patterns of NLRR1 and ALK at the mRNA level, which were not confirmed in clinical samples, although they exhibited no statistical significance. Taken together, NLRR1 and ALK were generally expressed in different populations of NB cells and exhibited mutually exclusive expression patterns mainly in immunohistochemical analyses.

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.

No MeSH data available.


Related in: MedlinePlus