Unfavorable neuroblastoma prognostic factor NLRR2 inhibits cell differentiation by transcriptional induction through JNK pathway

Abstract

The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)-mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA-induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c-Jun, a member of the activator protein-1 (AP-1) family in NB cells. Moreover, the expressions of NLRR2 and c-Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c-Jun reduced NLRR2 expression. We then searched AP-1 binding consensus in the NLRR2 promoter region and confirmed c-Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.

Keywords: JNK; NLRR2; c-Jun; differentiation; neuroblastoma.

Figure 1

NLRR2 is correlated with poor survival outcome in human neuroblastoma (NB) and regulates tumor cell growth in vitro and in vivo. (a) Kaplan–Meier survival curves. The average of the expression level was used as a threshold to divide the tumors with low (n = 55, blue line) from high (n = 23, red line) expression. High NLRR2 mRNA expression was significantly correlated with poor survival outcome (***P < 0.001). (b) Mock and NLRR2 bulk stable cells were generated in SK‐N‐BE cells and NLRR2 expressions were confirmed by western blotting (left panel). Stable expression of NLRR2 exhibited significant (**P < 0.01) enhancement of cell growth (right panel). (c, d) NLRR2 was transiently overexpressed in SK‐N‐BE and SMS‐SAN cells and confirmed by western blotting. Overexpression of NLRR2 significantly (*P < 0.05) enhanced cell growth in SK‐N‐BE (c) and SMS‐SAN (d) cells. (e, f) In vitro knockdown of NLRR2 reduced cell growth in SK‐N‐BE (e) and SMS‐SAN (f) cells. (g, h) SCID mice were subcutaneously inoculated with 1 × 10⁷ SK‐N‐BE cells. Two weeks after inoculation, when the tumors had an average volume of 70 ± 30 mm³, 1 nmol of control siRNA or a mixture of two siNLRR2 siRNA with 200 μL of atelocollagen was injected to the tumor mass to evaluate the growth inhibition effect. In vivo knockdown efficiency was measured by quantitative PCR (g). siNLRR2 treatment significantly (**P < 0.01) reduced the tumor growth compared with the control siRNA‐treated group (h). (i, j) Four weeks after treatment, the tumors were removed (i). The tumor weight was significantly (**P < 0.01) reduced in the siNLRR2‐treated group compared with the control siRNA group (j).

Figure 2

NLRR2 knockdown cells are susceptible to retinoic acid (RA)‐mediated inhibition of cell survival and differentiation. (a, b) SK‐N‐BE cells were transiently transfected with control siRNA or siNLRR2 and cell growth was measured using a real‐time cell imaging system. Knockdown of NLRR2 expression, as well as the effect of siNLRR2 on other NLRR family members were confirmed by quantitative PCR (a). The inhibition of cell growth by RA treatment was significantly increased in NLRR2 knockdown cells (P < 0.01). (c, d) SK‐N‐BE cells were transiently transfected with control siRNA or siNLRR2 followed by RA (2.5 μM) treatment for 3 days. The percentage of differentiated cells per field was counted (5 fields per well, 5 wells in each group). Data are presented as the mean ± SD. (e) SK‐N‐BE cells were transiently transfected with control siRNA or siNLRR2 followed by RA treatment for the indicated time period. GAP43, and neurofilament medium (NF‐M), markers for the neuronal differentiation, and NLRR2 expressions were examined by western blot analysis. NLRR2 knockdown cells with RA treatment showed a higher expression of GAP43 and NF‐M.

Figure 3

c‐Jun is important for regulating NLRR2 expression induced by retinoic acid (RA). (a, b) NLRR2 and c‐Jun were upregulated during RA‐mediated differentiation at the mRNA (a) and protein (b) levels in SK‐N‐BE, TGW and SMS‐SAN cells. Neuroblastoma (NB) cells were treated with RA (1.0 μM for TGW cells, 2.5 μM for SK‐N‐BE and SMS‐SAN cells) for the indicated time periods. NLRR2 and c‐Jun expressions were determined by RT‐PCR and western blot analyses. (c–e) SK‐N‐BE cells were transfected with control or c‐Jun siRNA (c and e, Santa Cruz; d, Cell Signaling Technology). Forty‐eight hours after transfection, expressions of c‐Jun and NLRR2 were measured by quantitative PCR (c, d). The data were shown as the mean ± SD. c‐Jun knockdown cells showed a reduced expression of NLRR2 upon RA treatment (e).

Figure 4

NLRR2 transcription is enhanced by retinoic acid (RA) through the recruitment of c‐Jun onto the promoter of NLRR2 while JNK inhibition suppresses the promoter activity. (a) Luciferase reporter constructs −790, +110; −560, +110; −315, +110; −147, +110 and −47, +110 (left panel) with renilla luciferase vector were introduced to HeLa cells and the promoter activity was measured by dual luciferase assay. RA treatment enhanced the NLRR2 promoter activity (−790, +110) (right panel). (b) RA treatment enhanced NLRR2 promoter activity in SK‐N‐BE cells. SK‐N‐BE cells were transfected with NLRR2 core luciferase reporter constructs (−790, +110) with renilla luciferase vector followed by RA treatment for 36 h and promoter activity was measured. (c) RA‐mediated c‐Jun recruitment to the NLRR2 promoter region containing AP‐1 consensus sequence was confirmed by ChIP assay.

Figure 5

The JNK pathway is important for regulating NLRR2 expression. (a) JNK inhibition reduced NLRR2 promoter activity in SK‐N‐BE cells. SK‐N‐BE cells were transfected with the (−790, +110) promoter construct and a Renilla luciferase vector. At 24 h after the transfection, cells were treated with SP600125 (50 μM) for 24 h and luciferase assay was performed. (b, c) TGW and SK‐N‐BE cells were treated with SP600125 for 24 h and the expression of c‐Jun and NLRR2 were analyzed by western blotting and RT‐PCR analyses. JNK inhibition by SP600125 suppressed the expression of NLRR2 and c‐Jun at mRNA (b) and protein levels (c) in TGW and SK‐N‐BE cells. (d, e) JNK activation by sorbitol treatment (300 μM) in SK‐N‐BE cells induced NLRR2 expression, which was blocked by SP600125 pretreatment. Total JNK, p‐JNK, total c‐Jun and p‐c‐Jun were examined by western blot analysis (d) and NLRR2 mRNA expression was determined by RT‐PCR (e).

Figure 6

NLRR2 expression is induced by CDDP and retinoic acid (RA) and contributes to RA resistance in neuroblastoma cells. (a) SK‐N‐BE and SMS‐SAN cells were treated with CDDP (10 μM) for the indicated time periods. The expressions of NLRR2 mRNA were measured by quantitative PCR. NLRR2 expression was induced by CDDP. (b) SK‐N‐BE mock and NLRR2 bulk stable cells were treated with RA and cell survival was analyzed using a real‐time cell imaging system. NLRR2 bulk stable cells showed significant resistance to RA‐mediated cell growth inhibition.

Figure 7

JNK inhibition in the cells overexpressing NLRR2 rescued the resistance phenotype to retinoic acid (RA) treatment and induced cell differentiation. SK‐N‐BE cells were enforced to express NLRR2 and treated with or without SP600125 followed by RA treatment. Cell growth (a), differentiation phenotype (b) and percent (%) of differentiated cells (c) were analyzed. The levels of p‐c‐Jun, NLRR2 and c‐Jun were determined by western blot analysis (d).

Figure 8

Schematic diagram of how NLRR2 contributes to poor prognosis of neuroblastoma (NB). Retinoic acid (RA)‐mediated JNK activation in NB cells induces NLRR2 expression that, in turn, ameliorates the differentiation and enhances cell survival.